Document text
1 Ii
Rev. 01-31-5003!+- A
FEDERAL BUREAU OF INVESTIGATION
Precedence: ROUTINE Date: 05/24/2005
To: Inspection Attn: I I
Washington Field Attn: SSA
SSA
From: Washington Field Office
Amerithrax-3
Contact = SAI I
ALL INFORMATION EBNTAIHEDAPP==°"ed B1" |:| 1' I FY
Drafted By: I I
V
I
MAJOR
Synopsis: To
environmental
December 2001
IVINS statedTitle: AMERITHRAX;Case ID #: 279A-WF222936USAMRIID* Pending!*92E£$92
CASE 184
summarize the events surrounding the unauthorized
surveys conducted by BRUCE§IVINS at USAMRIID in
and April 2002, and to provide an assessment of
motivations and documented actions.
Details: Following is a synopsis of the events surrounding the
unauthorized environmental surveys conducted by BRUCE IVINS atthe United States Army Medical Research Institute of Infectious
Diseases USAMRIID! in December 2001 and April 2002. IVINS'W'
September and
examination.surveys occurred after the anthraxlaced letters mailed in
October of 2001 were received at USAMRIID for
In the aftermath of IVINS April 2002 survey,
multiple investigations were initiated. The U.S. Army launchedboth Army Regulation ISE6 AR 15-6! and Criminal92Investigation
The
comprehensiveCommand T@IDT*investiga ions; Additionally, the FBI conducted anindep n ent query into the finding of Bacillus anéhracis B.a.!
contamination in "cold" non-containment! areas 0 USAMRIID I
pursuant to the AMERITHRAX investigation.
overriding purpose of the compilation and summary
contained herein was to provide a rigorous evaluation of IVINS
stated motivations and documented actions in relation to the
unauthorized sampling missions. Information from the AR l5~6,
CID and FBI investigations was compiled in order to construct a
summary of the events leading up to and in response
to IVINS survey results. Interviews and sworn statements
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ZHREIM In §N1LAaaIFEED
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derived from multiple USAMRIID employees were assessed for
details related to IVINS' actions, statements and motivations.
The individuals referenced herein werethe time of the environmental surveys. I I
were provided in response to Federal
Grand Jury subpoena #1228.
Writer's comments, enclosed in brackets and written in
italics, have been included throughout the summary. Comments
include observations regarding behaviors of USAMRIID employees,
notable supportive and contradictory information, comparisons of
results from multiple environmental surveys, and speculations
regarding possible alternative explanations for IVINS' actions.
The writer's comments are topics to be addressed during pending
interviews.
EVENTS PRECEDING IVINS' DECEMBER 2001 INDEPENDENT ENVIRONMENTAL
SURVEY
0 Concern regardin safe handling of B.a. EicAccording to[:::%:::::::::::]USAMRIID received the
Daschle letter for analysis on either October 15 15, 1
as initiall rocessed in Id saw exit w ' neg ove an carrying a slide in a small zi lock big. iiji77:Twasconcerned about contamination because[:fjhad assumed that[;:g::::]had forgotten to take off the love prior to exiting the
a oratory to the outside hallwav.2 I Ishargd thisI with BRUCE IVINSI I ||
samples were being processed initiall in 'oSafety Level 2 BSL-2! laboratory. According to E::E:E2E?i§jdid not raise this
concern as a huge issue. Additionally, was not scared, nordid[::]fee1 that other people's safety was threatened.* bx
,._";;-<During interviews with the FBI, IVINS[:::::::::::] L'V
stated independently that in the fall of 2001, after USAMRIID
received the Daschle letter tainted with B.a.,
[::::::::]concern that some of the handling procedures in the
Diagnostic Systems Division DSD! appeared to be unsafe.5
According to IVINS,[:::::]repeatedly voiced reservations
reqardinq the wav that incoming samples were handled during
I II Itold IVINS [:::::::::::]on
several occasions hat safety and contamination requirements were
not being met by ersonnel who were inventorying and
examinin eviden 7 b h " ce. was unsure a out t e specifics ofE;:::::::icomments, but indicated that statements occurred
uring normal office conversation. To it was not clear
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whether procedures were really not being performed safely, or be
whether procedures were just bein conducted differently than bqc[:::::]had seen previously, and|UiUUUUUinterpreted the
differences to be unsafe.9
Sometime soon after the qlove incident occurred,E::::::]
I U Iwas familiar with
the incident,I IIthe qlove was never in the suiterrather it wasI
tube of a smallLliquid Ehat accompanied Ehe Dascle lefter do carry to another
laboratory. The tube was double bagged in ziplocks and the r was bleached. L1Ejfiiiffelt comfortable with the fact that there were no
contamination concerns. [::::::]was not absolutely certain, but
I I relayed to IVINS that I:I
original concern about unsafe handling of evidence was no longer
a concern.
[WRITER'S COMMENTS Relative CO IVINS I
described very different reactions toI Isafety concerns.nterviews and in sworn statements,[::§::::::::::::]E?i7ii:jminimized the discussions of and inci ents related to
safety concerns, while IVINS inflated them. I I
referenced one or a few discussions, while IVINS indicated thatE::::::::] ' " ily" discussions re ardin safet Fad-mHlIlDl+-di g 9 Yconcerns indicated during an interview with the
FBI that was no longer concerned with contamination issuesfollowing discussion with[;;::::::::]referenced the gloveincident on multiple occasions urin interviews and sworn
statements. Neith r IVINS mentioned that
informed them of discussion with or that
at hI:I . conveye 0 t at was no longer concerned ]
0 Handlin? of the Daschle letter in suite B3 by IVINS and
In the fall of 2001 soon after receipt of the Daschleletter at USAMRIID, IVINS[::::::::]conducted preliminary work
with the B.a. evidence. IVINS was tasked with assessing the
concentration of the anthrax powder in the Daschle letter.
IVINS entered the evidence into USAMRIID's suite B3, a BSL3
laboratory, through the B3 pass-thru box. IVINS stated that
the contents of the letter contained a powder that was unlike
anything he had ever dealt with previously. Upon examination,
the powder contained in the letter seemed to float easily in the
air. IVINS was surprised by the fineness of the powder and
stated that "it floated around inside the hood like dust in the
sunlight." IVINS stated that due to the ease at which the
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powder became airborne, USAMRIID personnel should have conducted
testing within a laboratory that bore an overheard containment
hood.
IVINS DECEMBER Z001 INDEPENDENT ENVIRONMENTAL SURVEY
v IVINS motivations for conducting an unauthorized
environmental survey
IVINS became concerned that the fine powder of the
Daschle letter, as well as other potential anthrax letters and
samples, were not contained adequatel with the practices used atUSAMRIID. IV ' ed withI:LI processing of the Daschleletter in RoomEff:fififfj as the BSL2 laboratory was not
adequate to contain aerosolized B.a. powder. According to
IVINS,E:::::]commented "daily" to him that[::]employee
laboratory practices were unsafe. Based on conversations with[::;::;] IVINS became concerned about possible contamination
ou s1 e the actual laboratories where the evidence was being
handled. IVINS was also apprehensive that contamination of
personnel might lead to contamination of areas outside the
ainment suites. [::::::]mentioned to IVINS the concern thatEffimay have been exposed to powder and possibly contaminatedinOver time, IVINS concern extended to the office area which E::]
I I He felt that if there had been unsafe
handling of evidence, then[::::::]could have inadvertently
contaminated the office.
In December 2001, IVINS noted that[::::::::]entire desk
in room[::]of Building 1425, was quite dirty. IVINS described
E::]area as covered with an enormous amount of dust, debris and
other material. Accordin to IVINS, other areas in the office,
including IVINS desks were not similarly dirty.
Because of the condition of desk, and because he knew
that[::::::]had worked a great deal with B.a., both in his
laboratory and in support of Operation Noble Eagle, IVINS
wondered privately whether any of the dust and dirt on[:::]desk
contained B.a. spores. According to IVINS thinking, if a
spore was to fall in a dusty area, which had not subsequently
been cleaned, the spore could still be present.
Therefore, in December of 2001, tggk the[independenE:iitiative to swab the officel I IVINS specified the following reasons as to why
he decided to conduct random environmental sampling within his
office around.E::::::] desk area: his experience with the powder
in the Daschle lettery E:::::::]comments of possibly being
exposed [::::::]being administered antibiotics, and informationthat[:::]personnel did not utilize safety precautions.
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0 IVINS December 2001 microbiological survey
According to IVINS, he personall and privately decidedto obtain environmental samplings of [::::fi:]1desk, plate them on
Tryptic Soy Agar TSA!, and look to see if any colonies appeared
that were consistent with Bacillus species. IVINS swabbedapproximately 20 areas of[::;;:::]desk, including the telephone,
computer, and desktop. The p ates were taped, taken into the B3
suite and incubated overnight. The following day, the plates
were examined for colonies that had a colonial appearance
consistent with Bacillus species. Approximately half of the
plates yielded one to two colonies that suggested Bacillus
species were present. The presumptively positive colonies were
not examined by gram stain, nor were they examined for the
presence of the plasmids pXO1 and pXO2. IVINS did not examine
the colonies on capsule agar.
autoclaved and disposed of all the samples. IVINS
IVINS [::::::] of his findings, but was unsure if he actually
showed the samples. Because the possibility existed that
desk contained some B.a. s ores in the dust and dirt,IVINS decided to thoroughly clean[:?:::::] desk area, to include
everything on the desk. During one interview with the FBI,
IVINS stated that he placed the computer keyboard and wrist pad
in the pass-thru box to decontaminate them. IVINS did not
mention these specific actions during other interviews and in
sworn statements. IVINS in ' at he took no remedialmeasures beyond cleaning updE¬fif?:ftdesk because he did not
consider the level of contamina ion o be significant. He did
not believe that a full decontamination of the room was warranted
based on the finding of a few presumptively positive spores.
IVINS did not perceive himself or his coworkers to be at risk.
IVINS rovi l f bb p v ra reasons or swa ing only[::::::::]desk area. desk contained a laver of dust nopresent on either IVINS or desk. I '
5 | . Additionally
the airflow in room| |placed His| |desks upwind from
desk. |:|
With the exception of[:::::::] IVINS told no one at the
' his swabbing efforts and findings. IVINS did not advise
about the survey of E::]workspace, nor did he reveal to
the finding of presumptive positive colonies. IVINS
provided multiple reasons for not reporting his actions.
Because he was concerned that records might be obtained under the
Freedom of Information Act, IVINS did not keep records, nor did
he verify the presence of B.a. on the cultures. If the
colonies were not B.a., IVINS felt he would have been agitating
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many people for no real reason. Additionally, IVINS had no
desire to cry "Wolf!" simply because he had found presumptive
colonies that were not demonstrated to be B.a. or even Bacillus
species. If the colonies were B.a., he felt that he had
' ely taken care of the contamination issue by cleaningEfffffifldesk. IVINS reasoned that since USAMRIID personnel
were extraordinarily busy at the time processing samples for
Operation Noble Eagle, he believed that telling people of a
potential breach of containment and that B.a. spores were
inadvertently taken into noncontainment areas would have served
no beneficial purpose.
IVINS acknowledged, in retrospect, that although his
concern for biosafety was honest and his desire to refrain from
crying "Wolf!" unnecessarily was sincere, he should have notified
his supervisor ahead of time of his worries about a possible
breach in biocontainment. IVINS thought that quietly and
diligently cleaning the dirty desk area would both eliminate any
possible B.a. contamination, as well as prevent unintended
anxiety and alarm at USAMRIID." IVINS indicated that he did not
seek permission to conduct the survey, and he did not voice his
concerns about a possible breach in containment because he felt
that] Iat thetime, would have been dismissive of[:::]concerns.
[::g::::]confirmed that IVINS had informed two days
after he con ucted an environmental survey of esk areain December 2001. IVINS toldi::;::::]that he had swabbed| |
' ecause he was rne a out the comments made byEiEii?f:f IVINS advisedtiffjthat B.a. appeared to be r sent insome of the samples. During an FBI interview,[:::::?:indicated
that[:::]was not concerned by IVINS news because it could have
been anything, and[:::]was vaccinated.
[WRITER'S COMMENTS - IVINS justifications of his actions
following the independent sampling contradicted his explanation
of motives for conducting the survey in the first place. If
truly motivated by a concern of contamination on the coldside of
USAMRIID, upon evidence of such contamination, why did IVINS not
pursue more extensive methods toward correcting the problem? How
would improper handling of B.a. samples be corrected without
passing along information that the current containment system was
not working properly? Why even conduct the survey in the first
place if he did not want to cause alarm, especially if he
suspected that he would find contamination? IVINS had the
courage to conduct the swabbing without command approval, yet
lacked the initiative to inform the appropriate authorities when
the results were_presumptively_positive? IVINS was obviously
concerned enough about possible contamination to knowingly
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violate USAMRIID protocol, yet at the moment his concerns were
validated, he took no actions toward addressing the problems for
the benefit of USAMRIID.
f motivated by a concern for [::;::] why did IVINS not EECinform of the presumptive positive resu ts from E::]deEf::::]IVINS f' d d ' ' ' con irme uring an interview that he did no ' mof his findings; however, reasons for not tellingJt:;Ef?Twere not
provided. The assessment by IVINS that the leve o
contamination within the office was not a health risk to himself,
Idid not diminish the significance of finding
contamination outside of the hot suites.
IVINS ave conflicting accounts of exactlyinformed[::::::iabout the environmental survey. In
l5-6 sworn statement, he indicated that he mentioned
that he was going to check[::::::::]desk in December
after he had done so that about half of the cultureswhen he
IV '
to
an to
were
suspicious for anthrax. During other interviews, IVINS stated
that he told|[::::::]of the swabbing after the fact.
attitude wit respect to the results of IVINS' December swabbing' 'on to expressing minimal uneasiness with
regard to safety concerns,[:::::::]presented a cavalier
results.]
SPILL INCIDENT IN SUITE B3 ON APRIL 8, 2002
0 Description of Apfil 8. ZQQZ. incident in suite[::]
b6 On April 8, 2002, removed
eight 2liter flasks containing B.a. spores from a shaker
incubator in room[:::]of suite B3. The flasks, containing
erythromycin-resistant erm+! B.a., were transport ' two tripsb"/C
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B3. put the flasks in the hood and noticed that the paper on a£fari:lo| aboratory in roomT?:ifof suite
the inside. oticed dried media on the sides of severaltowe tape the cap of one of the flasks was discolored onof the flaskIijgi2ff:jnotified everyone in the suite of the
otential exposure and bleached off the ' '
within minutes. I lwere sent by| I42to the ward for nasal swabs and evaluation.
b0 Results o kuite B3 environmental survey b$c| &nstructed| |to perform surveillancecultures o mu ip e rooms insi e t e B3 suite on April 9-11,
2002, to determine the extent of contamination resulting from the
spill. Surface contamination by B.a. was identified in several
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areas of the suite. Some of the positive cultures in the B3
suite were emr+, while others tested erm-. The ermresistant erm+! strain contamination followad[::::::]tracks in the
laboratory suite.
Approximately 31 colonies of B.a. were cultured from
the handle of the B3 pass-box inside the suite. The colonies
derived from the pa - not tested for erythromycinsensitivity becauseif:?¬f:?f?§%i:%?ff]did not access the pass-box
on the day of the laboratory accident. An erm- result was
obtained for a colony from the B3 break room where the pass-box
was located, indicating that the contaminating colon was notassociated with the A ril 8, 2002, accident. [;::;:E:;hasconcerned thatE::::::?]found contamination on t e atc of the
passthru box, as the area was the point of entry and exit from
materials into and out of B3. All areas with contamination
were cleaned with 20% bleach and were negative upon resurvey by
swab culture.
indicated that the level of contamination found
during survey of suite B3 was approximately the same as
present on many occ ' en routine surveillance was performedin the hot suite. 1:i??i:jadvised that[:::]findings did not
appear out of the or inary or a laboratory environment. Though
spores were located on the "hot~side" handle of the passthru
box sam ling inside the box was negative, which indicated to[:::;;::]¬hat a breach in containment from the "hot-side" to the
"co side" had not occurred.
Followi th t d 1 'ng e sui e-wi e samp ing, held ameeting with the[:::::;::%:::]Division to advise of findings
and to notify personne t at "periodic sterilit chec s" would bereinstituted immediately. According to[::::ji:] prior to the
April 8, 2002 incident, bacteriological monitoring of the BSL3
was not cond ct d t l . U t'l mb ' u e rou ine y n i a nu er of ears prior to
2002 weekl surveillance w , y as performed. believed that
the practice stopped when anthrax research diminished and had not
been reinitiated on a regular basis.
Contrary to| IIVINS felt that the total
intensity of contamination from| |suitewide survey was
greater than expected and also of special concern, since spores
were found on the inside pass-box latch, as well as on shoes and
articles of clothing in the hot-side chan e rooms. On Monday,April 15, 2002, IVINS suggested to[::::::?:Fhat they also swab
parts of the coldside for possible contamination. IVINS
suggested areas to sample, including his office, the coldside of
the passthru box, and shower shoes in the coldside men's change
room. [:::::::]did not understand why IVINS made this suggestion
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because there was no indication of a breach in containment from
the hotside to the coldside. IVINS indicated that he was
concerned about the passthru box because that was where B.a.
entered int B3 I dd' ' o . n a ition, he was concerned about an incidentrelayed ' [:::::]in approximatel November 2001. IVINSinformedtf:%i?:?Tthat in November 2001,[f:::::]saw a USAMRIID
scientis wearing a glove that had been in the hot suite and
' the Daschl sealed in a plastic bag. IVINS toldEfifffifkhat he and considered the incident to be unsafe
practice, and that rried about contamination issues.
IVINS also informed that in December 2001, due to the
aforementioned glove incident, he swabbed| P desk in the
office shared by IVINS, He read presumptivepositives on a couple QL-pIates7-nut-are-n6L do any further
testin to definitively determine if the spores were B.a. [:::::::¬]told IVINS that if he never confirmed the s ores wereB.a., then the issue was not worth talking about. £?:;:;::]
discounted the importance of IVINS results since e 1 not
consider the issue important enough to confirm the results.
[:::]felt that IVINS concerns were an overreaction to a
relatively minor incident. [:::::::]specifically told IVINS not
to conduct any sampling without first obtaining approval from
According to advised[:::]on April 15,
2002, that IVINS had approached about conductin a sam linsurvey on the B3 passthru box on e cold side. [fE:;:::E:::§::]
I strongly advised IVINS against con ucting any
sampling. did not think that IVINS would conduct
unapproved samp ing.
[WRITER'S COMMENTS - Interesting to note that in all of his
interviews and statements regardin the independent survey, IVINSnever mentioned conversing with[::%::::] nor did IVINS relay[:;;:::::]strong a ' against unauthorized sampling. IVINSma e no mention toTffff?ffjof his concerns regarding the analysis
of the Daschle letter and powder in suite B3, or his perceptionof unsafe handling of B.a. evidence by[:::;V.Despite being
advised against such unauthorized action, INS went forward with
his independent sampling mission.]
IVINS APRIL 2002 INDEPENDENT ENVIRONMENTAL SURVEYS
0 IVINS motivations for conducting additional
unauthorized environmental surveys
On April 11 or 12, 2002, IVINS became aware of the
' ation incident in suite B3. In addition, he learned thatEiiii?iThad conducted environmental sampling in the suite and
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found several areas with presumptive positives for the presence
of B.a. On the following Monday, April 15, 2002, after
thinking over the weekend and becoming increasingly moreconcerned about possible contamination immediately outside the [:1
suite, IVINS decided to independently conduct environmental
surveys in areas likely, in his mind, to have been contaminated.
IVINS acknowledged that he should have requested authority from
his superiors to conduct the surveys, but he was afraid that it
would have taken weeks before permission was granted due to "red-
tape." IVINS felt that he had a legitimate concern due to the
possibility of the area being contaminated.
0 IVINS April 2002 microbiological survey
Without approval or consultation with anyone in the
chain of command, IVINS swabbed a number of locations outside the
E:]biocontainment suite on April 15-16, 2002. On April 15,2002, IVINS surveyed the coldsid pasjiihju box and
the coldside men's changin room o suit In
addition, he again swabbed desk area. According to
IVINS he sampled the coldsi e 0 the passthru box because[;::;;:::;]survey yielded a presumptive ositive for B.a. on thean e o the passthru box inside the[f:]suite. IVINS swabbed
the cold-side men's c ing room because he was concerned about
shoes worn inside the suite tracking spores into the changing
room. IVINS reswabbe desk area because he was
concerned that since was still working in and around the
B.a. evidence,[::]nmy ave inadvertently contaminated the area.
He also wanted to check whether he had done a good job cleaning
the desk in December 2001.
On April 16, 2002, IVINS analyzed the results of the
previous day's survey and found presumptive positives for B.a. on
five of 25 samples. The positives were found on: 1! the molding
by the passthru box, 2! the men's changing room table, 3! the. . I .shelf over the sink in the men s changing room, 4! o helockers in the men's changing room, and 5! on top Qfliiiii
computer hard drive. Based on the findings, IVINS continued
his independently initiated swabbing survey by obtaining samples
from additional areas, to include his office desk area located inroonxtép the cold-side[::]pass-thru box area, and the freezerslocate in the hallway outside the[::]suite. The areas on which
IVINS concentrated were those areas of high traffic, such as a
computer keyboard, and those areas of very low traffic, such as
the top of a book shelf. Of the 31 samples collected on the
second day, IVINS identified approximately 11 which appeared to
be presumptive positives. The initial positives from the April
16, 2002, survey were found at the following locations: 1!
outside the passthru box on the window sill, 2! outside the
passthru box on the electrical box, 3! IVINS office on the
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shelf under the air vent, 4! IVINS' office behind the computer
monitor, 5! IVINS' office on IVINS' desk by the penholder, 6!
IVINS' desk on the penholder, 7! IVINS' office on the upper shelf
in a tray to the left corner air vent, 8! IVINS' office on the
metal folder holder on the top shelf to the right of corner air
vent, 9! IVINS' office, top shelf of desk, left corner air vent,
10! IVINS' desk, and 11! IVINS' office on the wooden bookcase
shelves to the left of the desk. Of the 11 presumptive
positives, polymerase chain reaction PCR! analysis later
eliminated numbers 1 and 6, indicating that the bacterial
colonies were not B.a.
[WRITER'S COMMENTS - IVINS' explanations of his motivations for
the April 2002 independent survey were contradictory to his
actions following the December 2001 surve . If IVINS continued
to be legitimately concerned that was contaminating the
office space, wh did he not inform of his previous swabbingresults, or givef:::]some guidance wit regard to safe handling
of B.a.? One of IVINS' previous arguments for not notifying
USAMRIID command of his December 2001 swabbing was that he
believed that he had sufficiently cleaned the ' ed deskarea; however, IVINS used the possibility thatifffiiijdesk was
not completely decontaminated in Dece s one of thereasons to justify further swabbing. ffij:iffi:Tsurvey of the[::]
suite yielded no indication of a breach in containment from the
hot-side to the cold-side. Why were IVINS' convictions so strong
regarding possible contamination on the cold-side, while the
concerns of other experienced researchers, su h an dE;;;%:::] were satisfied by the results of[::ii:ff%;;;;;;;Jwithin
0 Results of IVINS' April 2002 environmental survey
In total, 56 samples were plated by IVINS on April 15-
16, 2002. Of the 25 sites surveyed for the presence of B.a. on
April 15, five presumptively positive samples were plated onto
capsule agar, and all five produced mucoid growth after
incubation. Of the additional 31 sites surveyed on April 16, 11
presumptive positives were obtained. None of the 11
presumptively positive colonies were plated on capsule agar.
Colony counts from all but one swipe indicated a very low level
of contamination, approximatel one to three spores. A singleswipe from just outside the[::ipassbox yielded greater than 200
spores. On April 18, 2002, 12 suspicious colonies, isolated from[f%i?f' April 15-16, 2002, cultures, were provided to USAMRIID's
for B.a. confirmation via PCR.
v Responses to IVINS' April 2002 survey
On April 16, 2002, after viewing the culture results
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had nd presumptive pos' ' or the presence of B.a. outside
the suite. IVINS told that he intended
furt er sam lin in their office and asked if he could sampledesk area. declined the offer to have[::] areasampled. s ted that|[;;]did not become involved in
the issue because had other ings to do.
on April 16 2002to conduct[:::]
Jggggmgggling that IVINS needed to talk toEventually, IVINS informedl::;;;;; he foundive positives for B.a. outsi e o eg?ijsuite."Effff2??jindicated that he had no prior knowle ge of the
surveillance cultures of several locations outsideAccording to[:;::;::g who viewed the cultures, one
a "pure colony o a out 200 spores, which was not
significant amount. The other samples yielded one
col ni a eared "dirt " with oth 'pp y er organismto on A ril 16, 2002,[:::]was advised b P
IVINS had identified contamination outside the laboratory.the suite."
appeared to be
considered a
%o two
rding
that
reported this information to advised
that IVINS told[:::]about the swabbing after the fact.
as upset that IVINS conducted the sampling without going
through the appropriate channels for approval."
On either the afternoon of 2002 or themorning of April 18, 2002 IVINS told??fii:iz]about his samplingsurvey and the findings. [:::::::]became very upset with IVINS
and told him that he should have gotten a roval. According toIVINS, on April 18, 2002, he went to and told|:F|of thesampling survey and the findings. E::;::::]was also upse with
IVINS for sampling without authority. Likewise, USAMRIID's
command staff was ver upset with IVINS actions." According toIVINS,[;::;::::::::::angry reaction to learning of IVINS
inde en en environmental sampling confirmed IVINS suspicionsthat%:::::::]would have opposed the survey if IVINS had sought
preapproval.
According on April 17, 2002, during a
conversation between and IVINS, IVINS stated that it
would be interesting to see what was lying around the facility.
[::::::]took the statement to mean that IVINS was curious as to
what microorganisms would be found if a sampling survey was
BOconducted. a
[WRITER'S COMMENTS - During an interview on March 31, 2005, IVINS
claimed that the path he cho e wab was the path that theDaschle letter took fitm2E::fi:fftTto that through the passbox
in the wall of suite B3. Tb the n y, IVINS did not swabthe hallway or locations near[::::ff:ff?i Aside from the areas
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near the B3 pass-box and the freezers in the hallway outside the
B3 suite, IVINS did not extensively survey the hallway leading to
were obtained from the shared office s ace in room 8 sam lesB3. Of the 56 samples collected on April 15-16, 20 38 samples
P I | P
were collected from
from locations near
the hallway between
that he swabbed the
fact that over halfthe men's locker room, and 10 samples derived
the B3 pass-box and the tops of freezers in
the B2/B3 hallway. Based on IVINS' claim
path of the Daschle letter, and given the
of the survey samples derived from his
office, the following question could be posed: Did IVINS have
reason to suspect contamination in his office because he had
intimate knowledge that the Daschle letter was present in room
at some point in time?
IVINS expressed, during multiple interviews and in
sworn statements, his concerns regarding the unsafe laboratory
practices of DSD employees. As a justification for his
unauthorized environmental surveys, IVINS cited information that
DSD personnel did not utilize safety_precautions. However,
during neither the December 2001 survey, nor the April 2002
survey, did IVINS swab re s a sociated with DSD laborato 'ersonnel aside from[:f::i:::id k d Tiff:ff:]
because it allowed to "point a finger" at DSD for poorp , es . Accor ing to IVINS,inwardly seemed pljisedgmijh IVINS' finding of contamination
83laboratory safety proce ures. Notably, the majority of the
B.a. contamination identified on the cold-side of Building 1425
was in locations associated with IVINS more so than DSD.
11: 1|'.IVIN.S" d d t linlOrmed:| resu s o in epen en environmenta survey.
egarding IVINS' survey
AFTERMATH OFIII
IVINS' APRIL 2002 INDEPENDENT SURVEYS
On A ril 16, 2 ' esponse to IVINS' independentsurvey,g::;;:fjdirectedEEi::i?:Tto collect samples from around
the col -s1 e of the pass-thru box, where IVINS located spores.
[::::::]went to the a sthru and noticed mortar dust on theledge by the box. [:?:jassumed that the dust derived from
drilling above the area during renovation work. [:::]took a
sample of the dust, which later yielded negative results for B.a.
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contamination. Approximately thirty minutes to an hour after theinitial collection,[:::::E2ffTurned to the pass-thru box to
conduct further sampling. then learned from IVINS that he
had wiped down the area around the passbox with water. IVINS
stated that he did not use bleach because he did not w npanic anyone with the strong bleach odor. In total,[%:E:Ef:]
collected 16 samples on April 16, 2002, from areas near the B3
passbox and from the women's B3 change room.
On April 18, 2002,[:::::::]was instructed to survey
additional sites to obtain a preliminary assessment of the extent
of contamination away from the pass-box. An additional 51 swipes
were taken in the common areas around suites B2 and B3, and in
Bacteriology offices located in rooms[:::::::::::::} .A second
surface decontamination was performed in the corridor where the
B3 passbox was located. Locker surfaces in the B3 cleansidemen's change room and desk surfaces in room[;:]were alsodecontaminated. All of the plates derived rom[:::::::::]April
16 and 18, 2002, surveys possessed growth of mixed environmental
microbial flora; however, all of the cultures were negative for
B.a.-like colonies.
On the morning of April 19, 2002, a "town hall meeting"
was conducted at USAMRIID to discuss the incident which occurred
in the B3 suite regarding the spill by| land the
ensuing environmental surveys. Personnel were notified that a
large scale sampling survey would be conducted to determine the
extent of contamination outside the containment laboratories.
Shortly before the meeting, discussed IVINS
mental survey and the results
the discussion| |was Ehe first t* earned
ores were found on e computer keyboard in office.
was very upset that IVINS swa bed the office and did not
tell More importantl , upset that IVINS had foundspores and did not tell[:fE] did not think that IVINthought through his act of swabbing areas of the cold side. i;;:]
knew of no independent swabbing by IVINS prior to April 2002.
fHRIIERLS_CQMMENIS As_Qf Anril 95. Z? I I l i
It 1S unclear if,
or when,| Iwas ever notified of the December 2001 survey
results.]
0 USAMRIID-wide microbiological survey
On April 19, 2002, an extensive environmental survey of
USAMRIID's Building 1425 was conducted. Samples were collected
from areas throughout the building, including nonBSL3
laboratories, office areas, corridors, animal areas, maintenance
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areas, support areas and passboxes outside all laboratories.E3; Samples were acquired from the Building 1425 break room and 22. . . e ¢I
and OnApril 19, 2002, a total of 838 samples were collecJed from 86
sites within the building. A total of 107 non-hemolytic,
bacillus-like NHB! colonies were obtained from the 838
samples.
On April 20-21, 2002, personnel from the Center for
Health Promotion and Preventative Medicine CHPPM! of Aberdeen
Proving Ground, Maryland, conducted additional sampling. CHPPM
Survey Set #1 included the laundry center at the Jeanne Bussard
Center and the DIS equipment. A total of 58 samples were
collected during CHPPM Survey #1 on April 20, 2002. Five NHB
colonies were obtained from five sites. On April 21, 2002, CHPPM
Survey Set #2 included samplin in the following areas: Bl, theB2 B3 hallway, the assbox toi::::] the men's clean change room[::%::], and roonq:; A total of 113 samples were collected from
nine sites within e building during CHPPM Survey #2. Of the
113 samples, nine NHB colonies were obtained.
On April 23-25 2002, su lemental surveys of theBudget office, room|:|and room|%|were conducted. Of the 65
total samples collected, two sites yielded a total of two NHB
colonies.
0 Results of USAMRIIDwide microbiological survey
Of the 1074 samples collected during the environmental
surveys on April 19-25, 2002, a total of 123 NHB colonies were
isolated from 102 sites. The 123 NHB colonies were identified as
Bact #1 through Bact #123.% The NHB colonies were subjected to
determination of sensitivity to gamma phage and to PCR analysis
for the presence of genes for capsule and protective antigen
PA!. Of the 123 total NHB colonies, only two colonies were
confirmed positive for the presence of both B.a. capsule and PA
genes. Notably, the two positives were obtained in locations
where positive results were previously reported following IVINS'
independent survey. The two colonies, identified as Bact #115
and #116, were confirmed positive for virulent B.a. and were
later genotyped as Ames. Bact #115 was obtained during CHPPM
y Set #2 and was described as "C-99, cleanside change room,[%EfE locker top." Bact #116 was obta' during CHPPM SurveySet #2 and was described as "C-78, rm.tEfj bookcase, rt, inside
door."
On May 8, 2002, the genotypes of 27 B.a. colonies
derived from the surveys were reported. Two of the colonies
resulted from the USAMRIIDwide microbiological survey, while the
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remaining 25 colonies derived from IVINS' cultures of April 15-
16, 2002. Of the 27 colonies, 20 were typed as the Ames strain,
four were typed as the Vollum 1B strain and two were typed as the
Sterne strain. No product was obtained from the analysis of one
of the 27 colonies. The 27 colonies represented B.a.
contamination in onl three locations. Of the 15 coloniesobtained from room[jf] IVINS' office!, 10 were Ames strain, two
were Sterne strain, two were Vollum 1B, and one yielded no
product during PCR analysis. Of the six colonies derived from
the cold-side of the B3 pass-box, all six typed as Ames. Of the
six tested from the men's B3 change room, four were B.a. Ames,
and two were B.a. Vollum 1B.
[WRITER'S COMMENTS - Following is a summary of the environmental
survey results, incorporating IVINS' data with that obtained in
the follow-up surveys. Between April 15-25, 2002, approximately
1197 samples were collected from over 100 locations. Of the 1197
samples, 16 cultures tested positive for the presence of virulent
B.a. Two of the B.a. cultures derived from the USAMRIID-wide
survey, while the remaining 14 derived from IVINS' surveys on
April 15-16, 2002. IVINS plated a total of 51 samples, 27% of
which yielded virulent B.a. colonies. Only two of the 1141
samples, or 0.18%, derived from the surveys conducted in response
to IVINS' independent swabbing, yielded virulent B.a. colonies.
Of the over 100 sites surveyed in Building 1425, positive results
for the presence of virulent B.a. were obtained in only threelocations: the men's change room in[:::;l the cold-side B3 pass-box, and room [:1 Notably, these were e three sites originallysurveyed by IVINf::¬f:fhj 22 ' surveys, only the officeshared by IVINS, and iffifijtested positive for the
presence of virulent B.a.] - _
0 Comparison between IVINS' survey and the USAMRIID-wide
survey
When asked why he was able to find virulent B.a. when
the other swabbing efforts did not, IVINS explained that he
utilized a more aggressive and extensive method for collecting
samples. According to IVINS, he sampled a larger area,
especially hard to reach and dusty areas, and used more force.
He also ex lained that prior to the large sgale 'ng surveyby CHPPM, I'D I tooksamples outside the[:g1laboratory and subsequently cleaned the
area thoroughly with each. IVINS stated that he believed
there were additional areas that were contaminated; however,
everyone had cleaned their areas as a preventative measure.
IVINS stated that the random sampling conducted by USAMRIID
safety staff consisted of about 6-10 swabbings in high traffic
areas, such as the phone, desk, and computer. He felt the
technique was inadequate. IVINS asserted that he took
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approximately 80 swabbings from his office alone, from high
traffic areas, and other areas near ventilation ducts.
SPECULATIONS REGARDING IVINS MOTIVATIONS FOR CONDUCTING
INDEPENDENT SURVEYS
| lwho was the] g
in April 2002, discussed two possible reasons why IVTNS conducts
microbiological contamination surveys in the non-containment
areas without command staff approval: ! IVINS did not follow
appropriate standard operating procedures SOPs! while working
with material and thought he may have contaminated some clean
areas of USAMRIID, or ! IVINS noticed another individual not
following SOPs and wanted to bring the problem to light. [::::::]
did not believe that IVINS was covering up a B.a. contaminationtrail which may be detected by the survey, nor did[;:]believe
that IVINS was involved in any criminal behavior re ating to the
AMERITHRAX investigation.
E:::;;] suspected that IVINS decided to conduct random
sampling o co areas because of the contamination incidentconcerning] | I Lstated that the finding ofcontamination in the pass-box area wit in the laboratory prompted
IV tg test the exterior of the passbox, as well. With regardtoms djoffice in roon1[::] [:::::::]suspected that IV ducte sample testing due to thepossibility Ehffgqégiibecame contaminated while working ir1[:::]According to IVINS had heard a rumor that the peo le in[:::]did not follow safety precautions. IVINS feared that E:::::]
may have crosscontaminated due to lack of properprocedure by people fron1[::E¥;:::::::::]
[:::::]did not know why IVINS conducted random
contamination sampling of the cold areas, but indicated that he
may have gotten word of the glove incident with[:::::::]
[WRITER'S COMMENTS - During an interview with the FBI, IVINS
noted a long-standing political rivalry between the Bacteriology
Division and DSD at USAMRIID. IVINS stated that Bacteriology
Division employees felt ostracized and belittled by DSD
researchers who were reportedly loathe to consult Bacteriology
employees superior expertise. 2 One could argue that IVINS was
partially motivated to conducting the independent survey by the
desire to make DSD look incompetent with regard to the safe
handling of B.a.; however such an argument is weakened by thefact that, aside from E::i::::]desk, IVINS did not survey any
locations directly associated with DSD.]
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SPECULATIONS REGARDING THE SOURCE OF CONTAMINATION
The Findings of the AR 15-6 Investigation reported that
a single source of contamination was not unambiguously
identified. Contamination was attributed to inadequate
decontamination of the outside of shipping containers brought
into and out of suite B3 of Building 1425. Additionally, it was
speculated that opening of evidentiary material in suite B3
created conditions leading to contamination outside of ziplocks
used to transport material out of the B3 pass-box and led to
contamination of the pass-box areas. 3
J IHI That strain
was different from Ehe strain causing contamination of the
USAMRIID cold areas. The latter strain was nonerythromycin
resistant. [:::::] concluded that the contamination was caused by
two different sources of B.a. 4 .A relationship was not
established between the surface contamination within the
biocontainment suite and that detected on the outside adjacentareas. 5 E::;:::;:::::g advised that there was no way of
determining ow ong t e cold-side areas had been contaminated,
as no prior efforts were conducted by USAMRIID to identify
possibly contaminated areas.m6
IVINS suspected that poor housekeeping and lack of
environmental controls caused the contamination. He further
speculated that the contamination resulted from USAMRIID
undertaking the role of examining evidence from law enforcement
agencies, while lacking a policy or plan in effect to complete
the mission. According to IVINS, USAMRIID had never dealt with
B.a. affixed to a powder and the anthrax letter evidence may have
arrived at USAMRIID in an outer package contaminated with B.a.m7
Specifically, IVINS speculated that B.a. detected on the cold-side Building[:::]nmy have been contamination from the Daschle
letter.
With regard to the contamination found on his desk,
IVINS indicated that he would sometimes set containers in ice
buckets on his desk so that he would remember to ship the
material or to fill out paperwork. The containers would remain
on his desk for a few hours or sometimes overnight. When live
B.a. was received from outside of USAMRIID, IVINS never opened
the outer shi ing container in the office. When IVINS took B.a.to Building'[f%t] he would either take it over directly or storeit in the refri erator just outside of[;;:] until he was ready togo to BuildimgEi:::] Typically, he wou not stop by his desk.
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[::::::]advised that[:::]did not know how[:::]office orthe other areas, became contaminated with B.a. spores. [:::::t]
suggested the possibility that DSD may have received an item that
b5 was not containerized roperly and the container exterior wasb7C contaminated. ° [:::::f]also speculated that the contamination
outside of the BSL-3 came from the anthrax letters.1 Due to the
fineness of the po t USAMRIID personnel tested and cameinto contact with,Efff:EEjthought it plausible that individuals
may have inadvertently transported the spores from one laboratory
her and to cold or administrative areas. As an exam le[E?:f%?recounted the glove incident[::::::::::::] which[::fa I
suspected was against protocol. 2
speculated that the contamination outside theBSL3 deri;;;:;;;L outside of the laboratory. E;::::::]based[:::]
on the pattern of contamination near t e passbox.Efffiffnoted that material was frequently returned from Building1412 after aerosol chal [::] also referenced incomingsamples from USAMRIID'stfE?ffDugway, Bioport, and other outside
agencies. 3
[::::::;]offered no independent ideas as to where the
, amination ou side of the BSL-3 came from, but indicated that Eiffjheard other people's theor that contamination may have comefrom the Daschle letter. 4 l::%::::lrecalled that on one occasion
a letter that was identifie as con aminated with anthrax was
taken into suite B3 by IVINS, and that the letter could have been
the source of contamination within the cold areas of USAMRIID. 5
[WRITER'S COMMENTS - If the contamination derived in part from
the handling of evidentiary material, to include the Daschle
letter, then why was contamination not also identified in[:::]
areas, where the bulk of the evidentiary analyses were conducted
and where all incoming unknown items were received?]
b5 NOTABLE REFERENCES BY IVINS TO HIS INDEPENDENT SURVEYS
b'}C
IVINS referenced his independent surve s and theperceived backlash in multiple email messa es tdy
| n June 16, 2002,
at 9:IB .m. IVINS sent a ' " p , message to stating the
atmosphere around USAMRIID is so poisonous these days that it's
hard to get much of anything done. I'm still persona non _rata,es ecially with people in power, like| qj[::?::::]seems to bark at me about lots of tin s these days." On
July 7, 2002, at 9:04 p.m., IVINS sent| Ia message
thal II i.Spring is when I found the break in
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mutant B. anthracis onto guinea pigs in the vet med cold suites.
The pigs started dying. I cultured their blood and did a plasmid
screen and found they had died from fully virulent pXO1+, PX02+
B. anthracis. Animal caretakers had taken dead pigs and put them
into the cold room. There was a lot of local decontamination in
rooms, hallway, coldroom, but nothing was mentioned outside of
this place. I am now forbidden from being a "cowboy." I can't
think for myself, and I can't do anything without everybody up
and down the line questioning me about it. I'm sure it's
punishment..."
During an interview on March 31, 2005, IVINS indicated
that he had no concerns about competency as a laboratory
worker, and his decision to survey rk area was notinfluenced by any perception that 'gent in[::]
safety practices. 7 However, IVINS sen multiple email
messages expressing views regarding performance in the
laboratory which were quite contrary to his interv' ents.On August 20, 2001, IVINS commented extensivel onI Iperformance in the laboratory. I Ito make some
Sterne spores last week, and to have them ready for today. [:::]
didn't have any backup blood plates, and didn't inoculate the
cultures until Friday. Today when the Leighton and
Doi cu s were a total bust, s insi e or outside thecells.t::j made some L&D mediumT?:ff:fj and inoculated some Ames
culture asks, and they made spores ine. and I think thatmaybe[::::::] messed up the salts b?en_haninf a lot
of problems lately makin good spores. It seems rushesthrouqh the work so that? I
IIt's very discouraging
't even take the slightest of
On July 6, 2001, IVINS wrote "more
rhad not vortexed Ehe spore
preps enou h t out the clumps, and so had very erratic
counts. did a heat shock with no water in the beaker
holding e u es and hardly any water about 1/8 inch! in thewater bath...If I sav anvthinq about ANYTHING tol I I
I On April 29,2001, IVINS wrote I | I
' ' ' 1 calculations,I I
basically just a satis actory
employee..."
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contamination here at RIID and became very suspicious to
people. I wouldn't be surprised if they wanted to quiz you about
me. Putting together all of the things I've heard about the
FBI's suspect list, my guess is that I'm on it being a middle-
aged white male who has made anthrax spores!. I can't tell you
how unappealing it is to be considered a mass murderer..."
On June 1s, 2002, at 9=oo p.m., IVINS sent $151
message indicating that "the FBI people who talked to me rig t
after I found the break in containment and got crucified for it!
said they thought that my coming back to work after hours was
extremely suspicious. I don't think anyone has any idea how
peaceful and quiet it can be here after hours an come hereand just sit, or read, or get on the web. If[::f:jis being
bothersome I can go back into B5 or even into B3. In the
evenings, B3 may as well be Mars. I can just sit and think and
relax from everything.! Also, it's just a great time to get work
done without hassle. It used to be honorable and commendable to
work more than 8 hours a day. Now, it's suspicious..."
During an interview on March 31, 2005, IVINS stated
that the December 2001 and April 2002 independent surveys
represented the only two times he ever swabbed in the cold areas
of USAMRIID outside of the hot suites. According to IVINS,
during the early 1990s he conducted swabbing inside the hot
suites as part of USAMRIIDs routine environmental monitoring
program. IVINS conducted the routine sampling at the direction
of his supervisors. The information provided by IVINS on March
31, 2005, contradi ' ation contained in an email message
sent from IVINS to On July 7, 2002, at 9:04 p.m.,
IVINS wrote to oh, guess what? This is actually the
third time I've oun virulent anthrax on the outside of the hot
suite. The other two times were hushed . The first time wasin the early 1980s. I ulhad injected some
guinea pigs and killed them! with the Vollum 1B strain. The
used pans of bedding, blood, urine, feces and all, had to be
deconned out of the suite, but the autoclaves were not working.
So they decided to paraformaldehyde the bedding and ship it out.
After the "decon" I - without authorization bad! bad!! but with
plenty of concern checked the bedding for sterility. I plated
some of it out. The results came back after unimmunized cagewash
workers had cleaned the pans. The very top of the bedding was
sterile, but below the top layer it was quite contaminated, with
anthrax and other bacteria. We're just lucky that nobody in
cagewash got anthrax. I told people of my finding, and after
that, used bedding in pans was never deconned by paraformaldehyde
- it just doesn't penetrate. The second time I found -|unauthorized! - virulent anthrax ouiside the suite was whensupposedly injected avirulent
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IVINS also expressed concerns regarding[:::::::]
ro ensity for spreading gossip. On August 20, 2001, IVINS sent
b6 an email message indicatinqj I
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rhas a tendency to exaggerate and to
spread nast sip, rumors, etc. about people, and I'm oin tobe right od [radar for a lonq time, I fear..| |
_LMR1IER;f COMMENTS: Based on IVINS written concerns regarding
pro ensity to talk, one could speculate that IVINS didnot inforn2[ii[:::jof his unauthorized surveys due to a lack of
confidence in ability to keep the information confidential.
Nbtably, however, IVINS did not address either his conce ith¬:;;::;:]propensity to spread gossip, or his issues withifi:T
a ora ory_performance when asked directly about these issues
during an interview on March 31, 2005. ]
On August 21, 2004, IVINS contacted SSA
to advise of an article that was published the revious ay inthe Los Angeles Times. IVINS faxed to SSA[::::f::]an article
titled "Anthrax Leaks Blamed in Lax Safety Habits: Sloppy
investigation of 2002 breaches at a federal biodefense facility,"
and authored by Time Staff Writer Charles Piller. According to
IVINS, the article detailed the results of the environmental
surveys conducted at USAMRIID in April 2002. IVINS claimed that
the article disclosed information that was confidential to the
FBI. IVINS indicated that he was upset to see the information
made public. IVINS believed the following statement to be
confidential to the FBI: "Three different anthranrstrains two
infectious and one a harmless vaccine were detected outside
biosafety labs." 9
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ENDNOTES
L 279AWF222936302 Serial 1699
2 AR 15-6 Investigation Exhibit #55
. 279AWF222936302 Serial 1699
AR 15-6 Investigation Exhibit #55
. 279A-WF222936302 Serials 1379 and 1700
. AR 15-6 Investigation Exhibit #19
. CID Exhibit #7
. 279A-WF222936302 Serial 1379
. AR 15-6 Investigation Exhibit #20
10 AR 15-6 Investigation Exhibit #43
1L 279A-WF222936302 Serial 1699
ll 279AWF222936302 Serial 1700
11 279AWF222936USAMRIID Serial 1223
14 279AWF222936~302 Serial 1700
92 1i CID Exhibit #7
; 16 AR 15-6 Investigation Exhibit #19
IT CID Exhibit #7
,1& AR 15-6 Investigation Exhibit #19
19 279AWF222936USAMRIID Serial 1223
20 279AWF222936USAMRIID Serial 1223
2L AR 15-6 Investigation Exhibit #19 ExSum 18 APR O2, Bruce
Ivins!
22 AR 15-6 Investigation Exhibit #19 ExSum 18 APR O2, Bruce
Ivins!
21 279AWF222936302 Serial 1700
24 279AWF222936302 Serial 1700
25 CID Exhibit #7
26 AR 15-6 Investigation Exhibit #19 ExSum - 18 APR O2, Bruce
Ivins!; 279AWF222936302 Serial 1700, CID Exhibit #7; 279AWF
222936~USAMRIID Serial 1223
21 AR 15-6 Investigation Exhibit #19 ExSum - 18 APR O2, Bruce
Ivins!; 279AWF222936302 Serial 1700; CID Exhibit #7
2& AR 15-6 Investigation Exhibit #19 ExSum - 18 APR O2, Bruce
Ivins!
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29 279AWF222936302 Serial 1700
30 279A-WF222936USAMRIID Serial
3L 279AWF222936USAMRIID Serial
31 279AWF222936USAMRIID Serial 1223
33 AR 15~6 Investigation Exhibit #19 ExSum - 18 APR O2, Bruce
Ivins!
3% AR 15-6 Investigation Exhibit #19
3i 279A-WF-222936-302 Serial 1700
36 AR 156 Investigation Exhibit #19 ExSum - 18 APR O2, Bruce
Ivins!
31 AR 15-6 Investigation Exhibit #19 ExSum 18 APR 02, Bruce
Ivins!
3& 279AWF222936USAMRIID Serial 1223
39 AR 15-6 Investigation Exhibit #20
40 279A-WF-222936-302 Serial 1379
4L 279AWF222936USAMRIID Serial 1223
42 AR 15-6 Investigation Exhibit #14 and 17; AR 15-6
Investigation Findings
41 279AWF-222936-302 Serial 1701
44 AR 15~6 Investigation Exhibit #18
45 AR 156
46 AR 15-61223
1223
Investigation Exhibit #17
Investigation Exhibit #14
4T 279AWF-222936-302 Serial 1701
4& AR 15-6 Investigation Exhibit #17
49 279AWF222936302 Serial 1567
5Q 279AWF222936302 Serial 1567
5L AR 15-6
52 AR 156
53 AR 15-6 Investigation Exhibit #19
54 279AWF222936302 Serial 1567
55 279AWF222936302 Serial 1567
56 AR 15-6 Investigation Exhibit #17
' AR57. 279AWF222936302 Serial 1567;
#17Investigation Exhibit #12
Investigation Exhibit #17
15-6 Investigation Exhibit
24 |:|
To: Washington Field From: Washington Fiely
Re: 279AWF222936USAMRIID, 05/24/2005
58 279AWF-222936-302 Serial 1701
59 279AWF-222936-302 Serial 1700
60 279AWF222936302 Serial 1700
6L CID Exhibit #7
62 AR 15-6 Investigation Exhibit #19 ExSum 18 APR 02, Bruce
Ivins!
61 279AWF222936~302 Serial 1700
6% 279AWF222936302 Serial l700;AR 15-6 Investigation Exhibit
#7
65 AR 15-6 Investigation Exhibit #7
66 279AWF-222936-302 Serial 1700;AR 15-6 Investigation Exhibit
#7
62 AR 15-6 Investigation Exhibit #19 ExSum 18 APR 02, Bruce
Ivins!
68 AR 15-6 Investigation Exhibit #7
69 279AWF222936302 Serial 1700
70 279AWF222936302 Serial 1379
7L 279AWF222936302 Serial 1701
72 AR 15-6 Investigation Exhibit #12
71 279AWF222936302
74 279AWF222936302
7i 279AWF222936~302
76 279AWF222936302
1567; CID Exhibit #7
72 279AWF222936302
78 279AWF222936302Serial
Serial
Serial
Serial
Serial
Serial1701
1567
1567
1700;
1700; CID Exhibit #7
1218
79 279AWF222936USAMRIID Serial 1223
80 279AWF222936-302 Serial 1699
8L 279AWF222936USAMRIID Serial 1223
82 AR 15-6 Investigation Exhibit #7
81 279AWF-222936USAMRIID Serial 1223
84 AR 15-6 Investigation Exhibits #7 a
8i 279AWF222936302 Serial 1567
86 AR 15-6 Investigation Exhibit #23
82 AR 15-6 Investigation Exhibit #7
25nd #21279A~WF222936302 Serial
b6
b7
|:|
L!{_;To: Washington Figd From: Washington Fielp
Re: 279AWF222936USAMRIID, O5/24/2005
8& AR 15-6 Investigation Exhibit #7
89 279AWF-222936-302 Serial 1700
9Q 279AWF222936302 Serial 1699
9L AR 15-6 Investigation Exhibit #8
92 AR 15-6 Investigation Exhibit #11
93 AR 15-6 Investigation Exhibit #10
94 AR 15-6 Investigation Exhibit #9
9i AR 15-6 Investigation Exhibit #6
96 AR 15-6 Investigation Exhibits #3,4 and 5
9T 279AWF222936302 Serial 1700
9& CID Exhibit #7
99 279AWF222936302 Serial 1229
1OQ CID Exhibit #1
10L CID Exhibit #1
102 279A-WF222936USAMRIID Serial 1223
103 AR 15-6 Investigation Findings
10¢ 279AWF-222936-302 Serial 1229
105 CID Exhibit #1
106 CID Exhibit #1
101 CID Exhibit #7
10& 279A~WF222936~302 Serial 3306
109 AR 15-6 Investigation Exhibit #54
110. CID Exhibit #1 _
11L AR 15-6 Investigation Exhibit #55
112 CID Exhibit #1
111 AR 15-6 Investigation Exhibit #17
114 AR 15-6 Investigation Exhibit #20
11$ CID Exhibit #1
116 279AWF222936USAMRIID Serial 1223
11T 279AWF-222936USAMRIID Serial 1223
11& 279AWF222936USAMRIID Serial 1223
119 279AWF222936USAMRIID Serial 900; 279AWF222936 1A 5512
Washington Figd From: Washington Fiel
279AWF222936USAMRIID, 05/24/2005
27Zb6
7C
» ~4' .~ ~92v
1~ K 0 O
- FD-302 Rev. 10-6-95!
ALL IIJFIIIFEZETIEIN IIEl]5T['AIl~3ED
HEPQEII-I Iii? l_llJlILAI'SIFIEEi
-1- DATE 1.2-lE-EEICIB Eli EIIITEE4 UC
FEDERAL BUREAU OF INVESTIGATION
Date of transcription Q 6 Z 18 Z 2 O O 5
As previously reported, according to United Stated Army
j Medical Research Instit te of Infectious Diseases USAMRIID!documentation, BRUCE ED&§RDS IVINS was granted four hours of annu
leave on September 17, 2 01. Ameflthrax investigators obtained
IVINS time records for the periods ending August 11, 2001 throug
December 1, 2001. For the two week time period starting September
9, 2001 and ending September 22, 2001, IVINS time sheet record
indicates that he took four hours of annual leave during day two
week two of the pay-period ending September 22, 2001. Since the
pay period started September 9, 2001, day two of week two of the
pay period is September 17, 2001.
' Copies of the documents referenced above the original
copies have been previously submitted! have been attached to and
made part of this FD302.
be
137CBAN DI.e TH
al
h
of
Investigation on O 6/1 8 /2 O O 5 at Frederick, Maryland
File # 2 7 9AWF 222 93 6 -USA192/IRI ID - 92?3QAp Date dictated
This ocument contains ll611¢l' recommendations nor conclusions of the FBI. It is the property of the FBI and is loaned to your agency;
it and its contents are not to be distributed outside your agency. Z
ALL IIJFi}PiI-LATIZIEII IIONFAIITEZZI '
IERIIN IS Y_TNCLAS5IFIEI
DATE I2-1-5-ZKIIIS ET EDE24 IE BAW; I;P§,~"I?I - Y
EMPLOYEE ID ELK/GRP ACT ORG EMPLOYEE NAME PLT ROT PERIOD ENDING SEQ NO280445449 0000 N4GPAA BBJE 1,; IVINS BRUCE E 08/11/01 00163
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WEEK DAY TYPE E/H LST TEM NIGHT INJ STARTHOUR HOURS JOB ORDER NUMBER OTH HR SFT DIFF NUM TIME INIT
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MON MON ------------------ --
TUE TUE ------------------ --
WED WED
THU THU ------------------ -- ,b c
mu ' mu ------------------ -- b7-C
SAT SAT
CERTIFICATION: ATTENDANCES AND ABSENCES CERTIFIED CORRECT. OVERTIME APPROVED IN ACCORDANCE WITH EXISTING LAWS AND REGULATIONSFOR NON-EXEMPT ELSA I DID NOT SUFFER OR PERMIT ANY OVERTIME WORK OTHER THAN AS REPORTED WE THIS Q
AS AMENDED CONTAINS INFORMATION SUBJECT TO THE PRIVACY ACT OF 1974 ' I
. AUT1-I7 I - I
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WK1
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FOR NON-EXEMPT FLSA. I DID NOT SUFFER OR PERMIT ANY OVERTIME WORK OTHER THAN AS REPORT OR THIS _ ERIOD. I
CONTAINS INFORMATION SUBJECT T0 THE PRIVACY ACT QF 1974 HS AMENDED II w'"'I*"*-;I1IE ~-=
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EMPLOYEE ID ELK/GRP ACT ORG I EMPLOYEE NAME PDT ROT PERIOD ENDING SEQ NO280445449 0800 N4GPAA BBJB IVINS BRUCE E 09/22/01 00155
swn JON | em M101CA | mes worm | 0145 - 1630 | TDCANS SUN MON TUE WED, Tl-IR FRI SAT SUIII THR FRI SAT
wo1mIo | a|oo a|oo s|oo s|oo s|oo I a|o0 e|90 |ncro RGORG-0RGO - RGIO TYP/SFT rag 0 -GRADE ND I I I I I I I I
WEEK DAY TYPE E/H DST TEM NIGHT INJ STARTHOUR HOURS JOB ORDER NUMBER OTH HR SFT DIFE NUM TIME INIT I
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use low, comp ism |suN, 2m: Pun Inn E/H LV IMP/Lv
NR1 IN OUT IN OUT IN OUT NK2 IN OUT IN OUT IN OUT REMARKS:
SUN SUN ------------------ - -
MON Mon ------------------ --"run TUE ------------------ -- .
wan wan ------------------ --
THU THU ------------------ --
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SAT SAT
CERTIFICATION: ATTENDANCES AND ABSENCES CERTIFIED CORRECT. OVERTIME APPROVED IN ACCORDANCE WITH EXISTING L IS AND RE TION$FOR NON-EXEMPT ELSA. I DID NOT SUFEER OR PERMIT ANY OVERTIME WORK OTHER THAN AS REPORTED .
CONTAINS INFORMATION SUBJECT TO THE PRIVACY ACT OF 1974 AS AMENDED H IAUTHORIZED SIGNATURE
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CONTAINS INFORMATION SUBJECT TO THE PRIVACY ACT OF 1974 AS AMENDEDAUTR R »I I
EMPLOYEE ID BIIK/GRP ACT ORG EMPL280445449 0800 N4GPAA EBJB IVINS BRUCE E 10/20
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2NDE NAME PLT ROT PERIOD ENDING SEQ NOEMPLOYEE ID ELK/GRP ACT ORG EMPLOYE250445449 OBOO W4GPAA BBJB IVINS BRUCE E 11/03/O1 00156
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CONTAINS INFORMATION SUBJECT TO THE PRIVACY ACT OF 1974 AS AMENDED- - - - - . - -~-
EMPLOYEE ID BLK/G-RP
200445449 0000 W4GPAA BBJB IVINSBRUCEE
swn JON | 01200 MIOICA
WEEK DAY TYPE
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CERTIFICATION: ATTENDANCES AND ABSENCES CERTIFIED CORRECT. OVERTIME APPROVED IN ACCORDANCE WITH EXISTING L WS AND REGULATIONS
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CONTAINS INFORMATION SUBJECT TO THE PRIVACY ACT OI 1974 AS AMENDED
AUTHORI ED SIG E
gi
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mwmvmama IN ., - ~ I
FEDERALBUREAUCWWNVESHGAHON
Precedence: ROUTINE Date: O6/O7/2005
ALL I§1IFCl15lILi1TICII1I CCIIIIITAII-TED
HEREI31 IS LTIJIILAESIFEED
, _ 031$ 12-15-2000 BY 60322 UEFrom: Washington Field
AMX #3
Contact: I ITo: Washington Field
Approved By: I
Drafted By: I
Case ID #= 279A-WF222936USAMRII Pending!-Y3?
Title: AMERITHRAX;
MAJOR CASE l84
Synopsis: To summarize the investigation of prescription drugs
used by Bruce Edwards Ivins during 2000 and 2001.
Details: Investigation to date has revealed that Bruce§§dwards
Iyipsd Microbiologist, United States Army Medical Research
Institute of Infectious Diseases USAMRIID!, was prescribed
various medication during 2000 and 2001. Below is a summary of
the medication known to have been prescribed to Ivins during 2000
and 2001:
Celexa®, filled 11/16/00 0 day supplY!; 12/19/00 0
day supply!; 1/12/O1 0 day supply!; 2/O6/O1 0 day supply!;
05/02/2001 90 day supply!; 06/27/01 5 day supply! and 7/31/01
90 day supply!, is used to treat depression. Ivins was
attending group counseling sessions during 2000 as a result of
depression. He appeared to have refilled the Celexa®
prescriptions before the supply was supposed to have
complete. Ivins received a 45 day supply of Celexa® on 6/27/O1
when he had 33 days remaining on the supply filled O5/O2/2001.
Additionally, Ivins filled a 90 day supply of Celexa on 7/31/O1
when he had 11 days remaining on the 45 day supply filled
6/27/Ol.been
Augmentin®, filled 3 1/O1 dav supplvlp is used totreat respiratory infections. H
|:|§| ¢§v1.ec/B.M1.T.- DI-if TH
!
1- s
InTo: Washington Fgd From: Washington Fielp b6
Re: 279AWF222936USAMRIID, 06/07/2005 b7C
Cephalexin®, filled 10/18/01 0 day supply!, is an
antibiotic. On 10/6/01, Ivins cut his finger but did not seek
medical attention. Ivins feared the finger was infected and in
an electronic email to[::::::::::::] on 10/16/01, Ivins mentions
that he should "probably go see someone and start taking
antibiotics."
Doxycycline HYC, filled 10/29/01 0 day supply! is an
antibiotic. No additional information has been obtained
regarding this prescription.
Diazepam, filled 11/13/01 day supply!, is an anti-
anxiety drug containing
Valium. No additional information has been obtained regarding
this prescription.
In an email to[::::::::::::]on 7/30/01, Ivins
discussed the fact that he was taking Zyprexa®. Zyprexa® is used
on patients diagnosed with Schizophrenia. No additional
information has been obtained regarding this prescription.
2
$1
Q 9To: Washington Fi d From: Washington Fiel
Re: 279A-WF222936-USAMRIID, 06/O7/2005
Ivins acceded to a polygraph examination on 2/28/02 and
at that time Ivins advised the polygraph examiner that he was a
taking Celexa.
An extensive review of electronic mail archives
associated with Ivins is ongoing. Additional pertinent
information obtained from the review will be documented in a
follow~up EC.
00
3
R .J. 0
1 w »-~' ~ 1
, 279A_WF_222936_UéAMRIID _92q£§L ALLIWQRMJIMJEMHEUED
HREIH _S FHCLASSIFIED[::::::::::] DATE 12-l5~2UU3 B? E6323 UCbe
b7C
. 1
F__________Ihe_fQllQwinQ_innesti1ative steps were conducted bY
and started on O4/O5/2005 in
Frederick, MD.
visual depiction 85 the family tree for four generations. The
source of the information was The Warren County Ohio
Genealogical Society Obituary Records, The National Comprehensive
Report on BRUCE E. IVINS, http:L/www92classmates.comL search
results and Catholic Review.Magazine at www.caholicreview.orq
search results and email sources. : S genealogy was compiled to produce a
Dates lof Death for g. WJEQR 1vn921s and his wife MARX R._Ivi§s and T. RAN ' , LL IVINS acquired from Warren County
Obituaries.
Information regarding the [:::::::::::::] was compiled
from Warren County Paper obituary section.
' ' the brothers of BRUCE E IVINS;
QL_ _ LN as procured from email sources.
H h1 -school information for BRUCE E. IVINS and[:::;:::][::::::;::::%}as acquired from http://www.classmates.com.
I I info ' d from email
sources and fr ha om t for BRUCE E.IVINS.|As well as, date of birth information relatinq to BRUCE E
IVINS'92 r.1 1 ._t n .l
An internet search
art ' 'icle ment1oning| l
Attached hereto and considered as part of this document
are copies of the above mentioned reports.
1 }r._<£v Me v.gai@g92'5.0'-92 05 .w PA-BAHEDKETH
All I30'FI]F!}1E1TIEE~i El]!-HAIIED
HFEEIEO I13 IJTIILLALSEEFIEII
DATE 12-l5~2L|I?3 E¥'1' 150320 UE E111i01,*"I1§§f'l 1-I
BRUCE E. IVINS FAMILY TREE
4
4
b
c. Wilbur Ivins-Phannacist Mamlvms W ,§
Retail Drug Business 40years Died at age 82 ~'
Died at age 67 Lebanon, OH OD D 26 Aug. 1954
DOD 27 Jan. 1938
T. RandallIvins- _ 0
Died at age 80 ofCHF , M15 M95
RanIvinsPharmacy1950-71 Dledofmr
Mason andE1kMember DOD4970
DOD - 13 Mar. 1985
4 U I5
r. - - - - - - _--
| Bruce E. Ivins :
ASIDE: Bmce had his 35th HS Renunion summer of 1999
Lebanon HS1960-1964
Last Updated 05/ 2005
I ,92 I . _Q __ILLNESS IS FATAI. I
T0 SAUIJIIIRIIICII
Associated With Fashion?
' Shoppe Many Years; _-
Rites Tuesday
Following an -illness of several
months, Saul Hurwich, agedj 60,,
died Sunday _at Jewish Hospital in
Cincinnati where he had been :2
4 patient for several weeks. '_§
He had been associated with hi!iwife Ruby in The Fashion Shoppeihereezfor more than 35 years. -A;i graduate of University of Pittgi:. burgh. he was an attorney and"
practiced law for a time 1&1 Pitta-
= burgh. He was a. member of Debi
'anon Lodge oflhiékonn, Scottish
_Rite,and _Shrine in Cincinnati and
was paste::alted ruler o{- the Lab-7
anon-/' Lodge jof Ell:'s.".'. i-' Funeral services were'_he1d'.; ;3'g.;
p. rn. Tuesday -"at' -5Pn'ne'ril_E
Home in-'Cincinnr't.i w'ith_-_buria.1_=i1ffwan : Hills?-Tewish :<;e=ii¢:=ry.».-;?.";'1. B_esidé's _'IllS-<:_W'I.fE -_1Ke1.le1?e3brotheiuand
H'~It?hI15I$0l1,0tto
10 IUI16 1954 I
o,n¢;;
~""Siiccji_i1'11l5s"At-I 78I . i .
Fliinelral-aervices for OtI:o.~"H1it-
'chinson," aged T8, '0! 'Cl.a1-ks'vill . . ' ..°v
Route -1,5retired farmer who" died
at Miarg. Rest;_Ho{!1e _Sundéy;ljvere,held at 10:30 a. in. Wednesday at
-._Vale Funeral _Hom'e' ;'_;inrl'.Morrow
'_witl1'_ 'burial'?_in .;:Mainevi1la 23 Cemeé
tery,;He:hadZi_>i$e:11.-Yin'I-illzliealth for
id. Survmngg. are,.h1a.-=ynfe}92-Tressxe, 1_;Ronté ,1;-;-I ohiii o£-Blancheaterglqnte
92
-.ll Feb 1954
Broth'er'Of'L0cal '
", Resident Called
Russell A. Hutchinson of To-
ledo, brotlicr.-u._'_,1Ii-s. Ralph Lewis,
was in ii serious condition and on
his 9292'ay_to Phoenix, Arizorizi. Mr.
Lewis neconipanled his bruthi'r-in-
law but .}l_r. llutchinso co'uIdn't
continue his fight for life and in
New Mexico passed away on Sat-
u1"dn_92_ February ti, at 10 p.m. Mr.Lewis I3 am-1-ompnnyingzllie body
home. ax'i'ivipF W0dn".~':Iny. NQ
funeral m'rn_i1|;ernents have In-r.-nmade, ' l _
___,_..___Hutchinson, Russell
Hutslar, Esther _
2_'7 May 1954
LOUISIANA WOIIIAN
IS C_0lIN.'I'YS':.~ SIXTH
TRAFFIOVICTINI-._. _ ___
Killed Beneath Own Car
In Mason Wednesday
, _ Night '_ _
"slqq. pa>1:>ou>1 .123 eqgj,-.zoqn.1a[aa
death of the year was rE¢0ldB<i
Wednesday night .when a 56-year-
old New '-Orleans WODZIQLI was kill-
ed underneath'_.her' own bar in l-IN!
village of "Mason; - -
Police Chief Elmer wright, who
investigated, reported Mrs. E_sther
Hutslar was en route to Spring-
field to visit a nephew. She be-
came tired of driving and asked!
her conipanion, Mrs. Gertrude-
Haasef 52, New Orleans to relieve
her. They stopped near the QalI:-
more Funeral Home. By mistake,
Mra.I-Iutslar left the car in auto-
matic . drive, As she_ walked in
front. of the ',maehine her com-panion'stepped on the" Ioot ne-Warren ' county:L _sirth' trafi:c-
Hutslar; downand ran pver her.
In her eieitemen Mrs. Haase put
the_car- in ~.reverse and the car
p85SE_d over-_ her again.;Rushed toMerci Hospital in . Hdmilton, she
w1;;.reported dead on arrival.. -
I The body was removed to Little-
'tor'1 Funeral Home ilSprlng£ielo._
Her nephew- is -Iiuther Doughman
'mi%ziaupoi§=¢-eater-19_z meaty-
23, Irwin, Ansel
9 Dec 1954-I'*"'Iiv5TI-:=_':"h?,Q~I;': I l W-
. ' 5 -iv Le, . day _.;.-_:.- 1 :5 I-r:=~* . =._fFo_r ~Ansel-drvvm ~;I§=.~,. ;.______1- "-:'-.5-t.§:;;~;..-I."
faml Irwin, "r:ed,s'r, nllmnbvn
Mason resident; diedl>=Mdnday'- at
his home, following-,a'n .ll1neas',q1
one year. Ha had been associated
with Muerlnich Motors until _ra-_
cently. _,.»." » '/ Funeral services will be held at;
2 p. m.- Thursdayat GalllmoreiFuneral Home withrburial in RosalHill Cemetery; -- " _ - -
--Surviving are his wife, Lucille,
two daughters, Mrs. Eleanor. Mo-
ler, Fflorlda; Mrs. Pearl 'B1'!l"-
near Mason; two sons, Glenn and
Clifford Irwin of, Mason and I-
brother, Will Irwin o£~Ml8°I1-
Friends are asked to_ omlt'iloJv-
._ers:and contribute to~~th8 »wl1"
ren County Cancer Sogiety.
. Ivins, Mary Randall i
26 Aug 1954 " ' 1;",
p Serviees Monday!.. Imi. Mary Randall. Ivins,"'n§ed-l2, died Fridny night at ,Chan'ip~ion Rest Home lie!-e"af_ter, an, ex-
Itended*_illness. She 92_92'1!I.'~l._£l. life-longI1-esldent of Lebanon and the wi-dow of C. Wilbur Ivinbfjwidely.
known Lebnnoh "_' lruggist'._ A, l
3 I{'i1nerul'serviébs vv.eFe' held _Mdn-dny at the convenieheej o'_f'tli'e {kin
il_vo[t O:iwald"Fdnernl Iloine jivithll>u1'inl *in' Lebanon _Qemet_eryI 1
~ -Siirviving-_ ar"a_ son_,.;'-T... Randull -Ivins and? three grandsons.
, ,_ .. --. _ . . * . .> _ .. e» <.- ~-
1
»
Mabel Ann lvins '
1 A 0 rm _ =Mrs/MabeluAnn lvins, 84, died
Aug. 12 at her residence,
Lebanon, Route one. _
She is survived by six
daughters, Mrs. Grace Bannest
and Mrs. Dorothy sieler. both of
Lqckland; Mrs. Catherine
Jones, Mrs. Esther Carey and
Mrs. Martha Jean Dunn. all
three of Lebanon. and Mrs.
Helen liauck of Sardinia. Also
surviving are four sons, Charles.
Leroy and Clayton, all of
Leba.non,' and Edward oi
Lockland. Other survivors in-
clude 31 grandchildren and 22
great grandchildren.
Funeral services were held
Saturday at the Oswald Funeral
Home with burial in Bethany
Cemetery. .
. =:?0E.<..igzo
Jonnn.@nu*,z_e;ot44s
EaatStreet,Leban_g:,died p .1
Wednesday at _,-{Kiddie -Q
Hen-L-..a= *3-. =--;
and _,. - -, .:,
M We M BS5
nawmussu§g§;_=zzpf§55=
at Oswald Heine;
Friends may 5 t3 __9__
p.m. Friday. Inteiint will-'inLebanon Comets:-if-"= =
pm--n "Q, I
John E. James/4 osc I970
John E. James, 70, of 445 South
,East St., Lebanon, died last
Wednesday in Middletown
Hospital.
He is survived by his wife,
_Ruth; one son, Dick; -and a
_- brother, Herschel, all of
zifliebanon.
3.; Funeral services were held at
2 P-1'Tl- Saturday at aswaldFuneral H°me in LebanonB Ice'§§.l°=lry. ~°"'."~="9./ aw 1 V 7 OStell/a Jameson
Mrs. Stella Jameson, 93, of 111
Mound St., Lebanon, died on
Jan. 12 at Miami Valley Hospital
in Dayton. She was a lifetime
member of the Lebanon
Presbyterian, Church.
Funeral services will be
conducted at 2 p.m. today
Wednesday! at the Oswald
Funeralliorne with burial in the
Lebanon Cemetery.
Mrs. Jameson is survived by
two daughters, Mrs. Clark
Weamer and'Mra. Cecil Griest
both of Lebanon, one grand-
daughter, and three great-
grandchildren.
Harold B. Janney10 no/ /174
Funeral services and burial
for Harold Brndden Janney,
former Warren County resident,
were held May 7 at Dayton. Agef
70, he died there May 5. Sur-1
viving are the wife, Mary Sophia"
Heaghman Janney; a daughter,
~ . Barbara K. Sloan oi Oak-
wood, and five grandchildren. A
son James died two years ago.
Elsie Johnston
2 7. J ua Y /9 70
Elsie K. Johnston, 74; of Route
1, Lebanon, passed away July 18
at the West View Nursing Home.
She is survived by a daughter,
Mrs. Marlin Watkins, of
Lebanon; a son, Paul W., of
Winchester; and four grand-
children. -
Private burial services were
held at 10:30 a.m. this moming
Wednesday! at Oswald Funeral
Home, Lebanon, with interment
at Spring Grove Cemetery,
Cincinnati.
Henry Johnston
11 J4/Li I? 70
Henry Johnston, 59, Route 1
Morrow, passed away at Epp
Memorial Hospital July 21..
Funeral services will be held
Thursday at 2 p.m. at Vale
Funeral Home. withlnterment
at South Lebanon Cemetary.
Visitation will be today Wed-
nesday! from 6-9 p.m.
He is survived by a son James,
Route 1, Morrow; a daughter,
Marlin Kersey, Dayton, two
grandchildren; three brothers,
Charles of Clarkavilie, Jess of
Westboro, and Howard oi
Wilmington; One sister, Neva
Runyan, of _ Q ... " 0- Q I Q
H;--.1 . , . ,:-5;;- x I 92;h¬' '1 ' _
.Letcli'er Jolinsariiii sf-9 as :1 KServices foe llefcher Joanne;
71.of1262Helai!ie &~:,n~;o£tm,formerly of am s. Union
1*1iw92W='92=ro.~ will be haunt '=_;zpm. today at_.the'. * "
Funeral l{om'e,j_ivit'l1'-tlie "7,John Swint oldangl
- "_-"I' :_.,-hiw _._92 .3; '7
Middletown-Hoipital.--2 .-. e. .~P92Surviving _are hio.;'-viriiidvri,
Franklin, and Sal-nu¢_1=
Redondo Beech,. Calif.;_--abrother, Fred of Franklin-;-'.a
iiter, Mrs. Ollie "ofMoms Fork. Ky.; andifour
grandchildren. . _ ' . _
Joe S. Johnston
Funeral services were held
Monday for Joe S. Johnston, 78,
of 13 Wilson Road, Fairborn.
Formerly of Waynesville,
Johnston died Friday at his
residence. 22. 05'? /770
He was a retired employe of
National Cash Register and_a
member of the Kryptons
Kentucky Lodge, number 905 F
& AM.
He is survived by his wife,
Ollie; two daughters, Mrs.
Fiossie Payne of Phillipsburg
and Mrs. Amanda Carwiie of
Dayton; three sons, John W. of
Springfield, Chester L. of
Dayton and Denman G. of
Texas; one bother, Wesley of
Richmond, Ky; 12 grand-
children, seven great grand-
children and several nieces and
nephews.
Funeral services were held at
2p.m. at Seven Pines, Ky. Burial
was at Seven Pines Cemetery.
Stubbs-Conner Funeral Home
was in charge of arrangements.
Paul Elton JohnstonI 9 sod H70
Paul Elton Johmton of 909
Snider Road, Mason,- died
February 12 at Cluint Hospital in
Cincinnati. He was 74 years old.
Mr. Johnston is survived by a
sister, Anna McKlbben of
Dayton, and a brother, Homer
Johnston oi Snider Road.
Funeral services were held on
Saturday at 1 p.m. at the Shorten
Funeral Home in M28011. Burial
was in the Monroe Presbyterian
Cemetery at Nlcholaville.»nnben'rmamiv¢n¢y_n' * 7Gardens. ;_;. >%,!~@,5 la -1
RM M» Fa-rat" la
Lester Hulbert -
-.9" J I4 NE I9 35?
Lester J. Hulbert. 88, of 4999
Waynésville Road. Oregonia. died
Saturday. June 1, .at Grandview
Hospital. Dayton.
He was a retired carpenter and
a member of the Carpenters
Local Union.
He was preceded in death by
his wife. Abbie Alma.
He is survived by one daughter.
Sue Kilburn of Miamisburg; one
grandson; and one nephew. .
Funeral service was held Tues-
day. June 4, at the Stubbs-
Conner Funeral Home in
Waynesville._'§Tirlal was in
Evergreen Cemetery, West Car-
rollton.
Leaola Hursh' l'7~ A P R. J? 95
Leaola Hursh. 86. formerly of
Middletown and Lebanon. died'
Friday. April 5. at Bethesda
Hospital. Cincinnati.
She was preceded in death by
her husband Lewis Hursh Sr. in
1952.
She is survived by two
daughters, Elaine Jordan of
Darien, Conn.. and Mildred Crane
of Lebanon; two sons. Robert Lee
Sr. of Cincinnati with whom she
made her home for the past 24
years. and Lewis Hursh Jr. of
Springboro; i4 grandchildren:
and 18 great grandchildren.
Funeral services were held
Tuesday. April 9. at Vorhis
Funeral Home. Middletown. In-
terment was in Woodside
Cemetery, Middletown.
~ Randall Ivins
13- MAR. ' Ii 5-5"
T. Randall lvins. 80. formerly
of Lebanon. died Wednesday.
Marph 6. in Frederick. Md..
where he lived.
Mr. lvins operated Ivins Phar-
macy in Lebanon from 1950 to
1971. After his retirement. he
worked as a substitute teacher in
the Lebanon schools. He was a
member of the Lebanon Elks
Lodge 422 and the Lebanon
Masonic Lodge 26. -
He is survived by three sons.
Thomas of Middletown. Charles
of California and Bruce of
Frederick. Md.
Services were" held Monday.
March ll. at the Oswald Funeral
Home. Lebanon. Rev. William
Johnson officiated. Burial was in
Lebanon Cemetery.
Memorial donations can be
made to the American Heart
Association.~ 0 3°r 0 1 l gt. T _I - §/6_._;%gI_:nolcZ IamesqnFormer_Warren County resi-
dent Arnold W. Iames. 67, of Ket-
tering. died Sunday. Oct. 13. at
home.
Iames was a member of the
Lebanon Eagles Lodge and the
Lebanon VFW. _
He also was a member of the
South Park United Methodist
Church, Dayton. and the John
Durst Scottish Rite. He retired in
1984 from Alernite Co.. where he
was a district sales manager.
Survivors include his wife.
Catherine: a daughter, Barbara
Lincoln of Kettering: a brother.
Earl Emerick of I-lot Springs.
Arie; and two grandchildren. '
Services will be at 10:30 a.rn.
Thursday. Oct. 1-7 . at the Rout-
songFuneral Home. Oakwood
and Irving avenues. Oakwood.
with the- Rev._Wa.lter Custer of-
ficiating. "
Calling hours will be from 5 to
9 p.m. today Wednesday. Oct.
16! at the.funeral home, with
Masonic services at '7 p.m.
Burial will be in David's
Cemetery. Kettering. " - _
Lena Iorns// saz. /W5
Lena . lorns. 92. East U.S.
22-3. Morrow. died Tuesday.
Sept. 3 at Bethesda92Care Warren
County following a traffic acci-
dent.
Born in Lebanon. she was a_'
member of the Lebanon
Presbyterian Church and a
charter member of the Warren
County Historical Society.
Survivors include a son.
Herschel of Williamsburg. Ky.: a
sister. Lucy Settlemire of Xenia:
and one granddaughter.
She was preceded in death by
three sisters. Helen Chamberlain.
Etta Nixon. and Grace Middleton.
and a brother. Walter.
Services were held Friday.
Sept. 6. at the Lebanon
Presbyterian Church with the
Rev. William Johnson officiating.
Contributions may be rnade to
either the Warren County
Historical Society or the Lebanon
Presbyterian Church.
The Oswald Funeral Home.
Lebanon. was in charge of ar-
rangements.9! Slggllie Jackson 5if 1%.?!
Mollie Jackson. 97. died Sun-
day. Sept. 1. at the Quaker
Heights Nursing Home,
Waynesville.
Survivors include three
daughters. Gladys Ell/tins. Frieda
Petty and Fon Eva Brown; two
sisters. Matilda Owens and Nettie
Station: and 20 grandchildren
and 31 great-grandchildren.
Services were held Wednesday,
Sept. 4. at the Breitenbach
Funeral Home. Middletown.
Burial was in Woodside
Cemetery. Middletown.
Nora Jadwin' I 7 J um zq 8.5
Nora N. Jadwin, 94, of the Ot-
terbein Home, near Lebanon.
died there Wednesday. July 10.
A memorial service will be held
at the convenience of the family.
- Chauncey Jeffers W
X9 -. F155. I/'31
_ Rev. Chaunceyyli. Jeffers. 95.
of the Otterbein Home, near
Lebanon. died Wednesday. Feb.
13. at Middletown Regional
Hospital.
Rev. Jeffers was a graduate of
Taylor -University. Wesley
Theological Seminary. He was for
70 years a minister in the
Methodist Protestant, the
Methodist. and the United
Methodist churches. For the past
seven years. he was active tin
volunteer service at the Otterbein
Home.
He is survived by his wife. Ern-
ma Jeffers; two sons. Glen of
Fostoria and Harold of Chester
Hill: two daughters. Mrs.
Rosemary Osborn of Newark and
Mrs. Miriam Wagner of Dayton;
16 grandchildren and 14 great-
grandchildren. -
Memorial services will be held
Saturday. -Feb. 23. at the Otter-
bein Home Chapel at 2 p.m. Rev.
Wiiiiarn McOmber will oiilciate.
Burial ~ will .be in Dtterbein
Cemetery. "
' ¢
; ii -' __-»_s.{._.- éi 1,... '- ~- __ klin resi j ' J05§|;lll"[.?l?lIl. . 11.59-, °f
we Myers, Fla., iiiéd Kitties eveningij *1 H 5 '1 . . a'tg:fIlE}llfl Gltdr:l%?gfVR,oh§!?V-?6litE l as ex=y gcllliv secretary oiihe Building Trade. J Council oi Dayton. -In 1982. he retired'~ l 1}; .» aridmovedloFlorida. _ .' ii? ' are is survived by his wire,-lda; his
idther, Mrs. Margaret Hu_f¢h.1"5° M;',§zL~.- ,-Middietown; a son, Walter Thomas. oi. ':"~iimnkiin: one daugiiter, MF5- PeggyYolk bi'l'erre Haute Ind; °"¢b1'0¢h@l ."'°92 William oi Middeltown; W" sisters.
' Sister Ann Hutchinson of Indianapolis,
-I and Mrs. Mary Webb of Clearwater,
- = d h'id e . - .F¥il%1'as?sndi1{lv£l7%i1rls]tiz£nl- Brugial will D?,, / 7' L/y nniiunias :1 _ . _ 6 .1 e jtdilédll 1VIH5§/7/.'
0 / 5
. -1
,.
I
'- Wedriesday at 10 a.m. at the Holy Trini-
I
', 7" ii/l'ddletoWIl. the Rev-t §l,eo?;<-fllglelg otiliciating. Burial will
follow at Woodside Cemetery. _V92l5llHf1°I1
will be Tuesday from T to'9 p.in. at the
Wilson-Schramm Memorial Home in-Middletown. _ '_ ' b t,= _M¬lllOX'l3l contributions may 8 _$<-$11
to the American Cancer $0CletY=asbestos'research. ' '
' n . ' .. i.. !§.,t?t*r.§9.. /48:1ohnson 63. 2720 Fac I=.i.'~ ;. 1-
zél-ll-3 M
.19 R.<>.es1.- ..$rsiealie¥°¢-2.s11=¢
:' !1:*£"i;&.l - . - ~;r. -.i ;'~z. Ti ~-=~" -.-e .1'. t 573;}. tip? 1-1".élnative of Ken cky;
0-iv -. '0 v,i'}3f'¬_;»:,_ ;,£':_*' is .-'
two,Gie;i=ir.i.ituetoi1; of
.-EseenieI;taiiaI.reergaaiiaaiinareaia:-
11 as Sm?"
='.Eim¢1?el*..Horne._W¢$t C¬-1T°1lt°.!~-Rev- Delbert Dawes.0f-
i_'_3<':ia.t_ing.-.-_Burial was; in Spr-
lngtioro Cemetery. - - »
," Mr: Neal Johnson, 59, 23¢ Walnut
If Ave.,idied Monday, Nov. 18, at Ketter- ing lvlijedical Center where he had been a In; ?ElllE tfor two weeks. He had been ill , oralongperiudottime.'- -_ - v --;'.;'._- Mr - Johnson was born in -MadisonCounly, Kty.,- and had lived in the Carli-teisle aea or most of his life. He had work d as a foreman at the Valley Sheet
»:-Mei , Co. in Mlddletown. He held thejfrank of sergeant in the U.S. Army dur-I ing Iorld i ar ll and was a member of
Bethany Baptist Church. .iii 8 at Kefterin§~hilqd_i_081
,-:,_7_ ~ _ __J.::. ;_A 5...__;;-, 92_.,,.5
".2 - . .-L .4. -w .-.~-"- '-".i-~vgs. §1 rsd. fron.i.i2es?fPnrY92l=.},1tJP-°Y -u-, _ _-~: ,-.;.. _92,_-- _~»:- , .»-,- 1 I~'~ 4- t.» 2. .=- _ . ice .» at 1 ' » $3». 215'. -1' 4 '~;:"J5**T"?"5 '3 -*5 'i?§§$hr§or§" '"inEludE7.1 .11}?-i;I1-lliftif -1
.:i::.~.'i- _- ~' *-:.: ==.. .- . ' '1' J mfg- =5
f_mgb9m;2§_$veg};;.'sis _t'g;s;anglI;one;A
- at =, .,r».'Z- -_ -'1' '*-','§.;Funeia1_---services _were-L held ~
1 ¬_§Ne!al Johnson; *§§'§Z? hI
mo
SIégggai ve sods--reed 5htliTérry oi
i letown; his mother, Mrg Ramaii?! ii" °f Waw. Kim four brothers
I, ussell of Cl_earwater,.Fl_ andiii. "ii8iiil ii"'°¬ i *1 -W" Sires» 1Ky - 0| on of Nicholsville,,M1l_ton__ Ingles /A >3."
_ LEBANON =-= Milton lngles, 76 ofCelina, formerly of Lebanon, died athis home Thurs aiy.He was prece ed in death by his
wife, M. Jeanette, in 1983.Survivors include a daughter, Mrs.Jo Anne Adkins of Ce na, three
grandchildren and a great-
grandchild,"
Funeral; services will be at 11 a.m.glonday at the Oswald Fimeral Homeere.
Burial wiH f0l10W"al; Miami ValleyMemo Ga ' 'ry rdens.
Visitation will be Sunday from 2 to 5and 7 to 9 p.m. at the funeral home. ,
Fr? L0uis Jeffery ,,j.<;.r/: _
Skokiaan Drive. Franklin. died
Nov. 17 at Middlétown Regional
Hospital. ' _
He» retired from Stone Con-
_talner. Frankliniri 1975. He wasLouis Bud! Jeffery. so. er 44}
I
a veteran of.World War ll. . . .'
5 Survivors include his wife.
Phyllis: one son. Tracy. at home:
daughters. Mrs. Diana Love. of:
Webster. Fla. and-Mrs. Ineiebie
._Charies of flaijjisbiii" of
l?rice 'of92 Glendale, Calii'.; two
- granddaugl1ters."?'. _ ' _: _
Unglesbif-;'A.nderson_'F'ifiiérai_' l¢lome;.;1f<jif_a_1jilcli_ii. witli;,_tl_ie Rev.
James Maggard _ officiating.Burial 1-v.ilil~' be -in Spijinghoro"
Cemetery". Visitation will be
Wednesda Nov. 20 from 4 to 9
Hi "survivors include his wife
e and Kenny, Ted and Everett of I
°5 W311". Lawrence oi Glendale' Moore of Fianlilin}-llvga;'i§,Fotl i ers.'*
s i~ia.. Mr"s._ Margaret I_921viiii:;;i§i;ie_._o§,- Madison. Fla. and Mrs'..=:Clara"
'..
$@r'vi<:<=s:.-will be h<?l¢?¬T¥ieiS§1ay,¢-
,;,u<"=v- ,. 2 -p-m-E.-;at ithei.
p.m. at tl7§e'fun'eral homé.' ' Qi ,g:_gi¢iisie;*-Ltiritgd->aii;»i7r2£iiitiiii.-it
Robert and 1tI@Yi!=e119§h.!>.EB¬*-l.¥!1§'5<~é
§e.~_ Fla._:y inree*i"¢2_eisiej5a?.e§ii;si;%§;-Mildred? Morton " of '. Briadei'itoii.1'*LEBANON . T. Randall lvins,_ 80,
oi Frederick Md., aformerlon timeLebanontresldent, died Wednes ay at
Homewood Retirement Center in
Frederick where he had resided the
past year.
He operated the lvins Pharmacy
here from 1950 to 1971 and was a
substitute teacher in Lebanon schools
after retirement. He was a member of
the Lebanon Elks Lodge 422 and
Lebanon Masonic Lodge 26.
' He is survived by three sons,
Thomas of Middletown, Charles of
California and Bruce of Frederick,
and three grandchildren.
Services will be at 11 a.m. Mondayat the'Osw'ald Funeral Home. Burial
will be in Lebanon Cemetery. Visita-
tion will be Monday one hour before
' services at the fimeral home.
- .
Jeri-:§:?if/W F»35NKi.-' IN Ga A. Jones, 24, ciFfé_ll1k11i_1,- died Welilliesday. 31; inUniversity of Cincinnati Hospital of ;gégéflllglsrérigheiéuneral home woiilia .-. _Mr. Jones was ax-t technician .fag Reynolds and Reynolds Compntei5;. ' '_ l :;;*' ' fr '§':::'.i.- ;~iii: He issni-vived by his 92;:lf8,;§_3_l:3fll'3;ecggi ,'ln>y.; rather, Janiesigrqnee oir: I11-R=T*l1;< ._1i;qther._?_1i4rs-_.~.iiiei .A.
'-.-1 ' ¢..0ll ".' -['ga__ndfathei',Roy. ",_ 9? 39- -ire?
';"ei;ar:sergia"" fiivill _ri;'e"; iii"-"1'iii.isewraer-aner<?§esioe1¢?>iii§ir@i
;.§°4s1r:.i~Kemrerit1*§¢d:-t5nr~= dalee;1e2Rev.iJerrIx§;MueHe?;;of-3,-_£ic:ating. Burial will be l_tes,t'l-laven,§:_Cc§metm'";g gligétggon will -lieuat theF. _ __ o oi-e.sei;viees;jg:- 'I1¥l'!I45-ll§ial11_¢~l1_l8_ by_.:;tl;ie;.J{orhis§§§lineral_ uieinSpnngdale¥3_I§:;,~5':_-;is-=:.Contnbutions may be'made.to the92;§,ii'll_os'piceofhliddletown." .
. ii.-'t ""]éh'ti I'5auLi"ohes ~- 1,... .. i" -".*92:J¢'_.p'__I=§~l' Jig '- : g n Paul¬§J§m§§?;gf",of ioeo:D aléjAve.. Franklin. died Jan. 21
at Gréndview Hospital. Payton.
He_is survived by his wife, Bet-
ty; son John Jones of Dayton;
three daughters, Mrs. LindaDalton of Carlisle, Mrs. Susan
§_Ciaycraft of Franldin-.,-ai1_dLouise Hall of Miclilgan; _two
brothers. Arthur of Missouri and
Earl of Zanesvillei two--sisters,
Mrs. Rose Burden of McComb
and Mrs. Lucille 'Atrash of
~el5ii§'w. lgdgwre ~_'.... .'....l',:..Z , 92,'_-._ 1.1.r Elvis wflgo,-rs, 6f6_96_>5'Fran -
Madison Road, Mlddletowhfdied at
- -3-p.m. Sunday, May 5,1996, at his
i'esidenc":e., V ..§_.§,Bomln Mo're-__h§?.d~ KY-..0n ' 51'
Aprii7, 1923,he lived here - .
most of his_ r- life. Mr. lgo
xmemplovedin thefactory ._at-inland _Con- '
tainer Corp. for 1
32 years. He -
_ 1'etiredin- -1984; He was Mr go
_.:a member of the Full Gospel Out-
_;_rea'oh Pentecostal Church of ,God
_' Surviving are his wife of 53 years,
Q ;3arrie J1; three sons, Eddie LeRoy
l o of Franklin and Richard and Jef-.. Q
,frey lgo, both of Middletown; a
daughter, Linda S. Nolen of Franklin;
[eight grandchildren; two great-grand-
";children;'a brother, Harry of Toledo;
"and a sister, Betty Jane Cecil of
»Morehead, Ky. ~ ..
, He was preceded in death by three
'brothel_'s,'l_eonard,13uck and Roy lgo;
and a sister, Myrtle Carroll.
Y ARRANGEMENTS: Funeral ser-
vices are set for 2p.m. Wednesday
at the Joseph R. Baker Funeral
Home, with the Rev. James Maggard
officiating. Burial will be at Woodside
Cemetery. ' _ . .
Glqvsl-Iltla 1~*¥f-i .-lair _- F-._r;},1a,<5,1yg¬7I-lazel Gum! Ivins.
of.-Iieban'orr__for most of her
lifei..dIed'_lv_Idn_day.J@1I}- %_2~.' -,. '_borr1'ir'_i _1l I?"-f_1,iI_1E°'¥'l "
~w.i/at "*5Yiii4l*§i17£il?.9'f?._lPTi -t Y .D. _a1i¢wqo<1sie Frances Raw!' "Gi1:£,SHe;1rétiisd,in'l9_73~aeY
'29 as a secretary for the
=WafiIéri. County Board, l 0f
E3fi'c'=it5idn.an_d was a member of
tlie§.Eei§ahon' United Methodist
*'l:l:13l'1'v.c'lt1;':' and L the _Lebanon
Cblnifriltgfblub. . 2 ' _-.s1'1¢,wais preceded in death-
.by-heplarents; and husband
Charla?-°e.Q<5e=Z.5~. 1994-:
"5'i1~.'?/lié.t%.1§t~I@71?_ 1=§.£?1¥5"i ??i¥ ?"l@Y'
Jar1_r,25:<f.iT9m.-5'7;-.pZni. _withineralsérvice Friday. Jan. 26
at. 10,, a..m. . all? at Oswald-
Hoskins Funeral Home.
Lebano1_1"with Rev. James
Ludwick ofciating. Burial will
follow in Lebanon Cemetery.Freda B; Inloes. ,?1~'*.=B.»- r@9¢
CHAUTAUQUA Mrs. Freda
Blanche Inloes, 80, of 10041
Elanja Drive, Miamisburg
Chautauqua!,'died at 7:10 a.m.
Thursday at the Barbara Parke
Care Center in Middletown.
A homemaker, she was born on
March 1, 1915, in College Cor-
ner, Ohio, and formerlyresided
in Oxford. -
Mrs. Inloes is survived by her
husband of 54 years, Louis_H.;
three sons, Bob of Faireld, Ed
of Carlisle and.. Geraldof San
Antonio, Texas;' a_ daughter,
Martha L. Reese of Miamisburg;
two sisters, Hilda Agnew of
Eaton and Patty Master of Ox-
ford;. seven grandchildren, Juli
Reese, Diana Reese Clary and
David, Nichole, Jerry, Stephen
and Christopher Inloes; and two
great-grandchildren. ,'
She was preceded in death by
-two grandchildren, Susan Reese
in 1992 and Brett Inloes in 1977.
Funeral services will be at 2
p.m. Saturday at the Unglesby-
Anderson Funeral, 1357 E. Sec-
ond St., Franklin, with the Rev.
Ted_ Holstein ofciating. Burial
will be in Butler County Memo-
rial Parkf -
4Charles lnnis 1'1 l=sB- /9%
Charles Franlclin 80, of
Kings Mills, died Monday.'Feb. 19"
at Bethesda North Hospital. '
_IjIe was born in 19-15 in
Maineville and had been employed
in machine repair with Ford Motor
Co. He was anArmy veterarf. '
__He was preceded in death by
his"parents Lulu Mae Kendle] and
. Lucian '
He is survived by wife Miriam
Boger! Innis; sisters Mildred
Holden of Urbana and Janet
McDaniel_ of Lebanon; brothers
John of Dayton, Robert of Blue
Ash-and Lester of Montgomery;
andmany nieces and nephews. "
' Funeral service was held
Wednesday, Feb. 21 _at 1:30 p.m.
at Tufts Schildmeyer Family
Funeral Chapel in Loveland with
burial at Hopktinsville Cemetery.
' *5 .7 J}? N James Innis /ea-sf
James Robert Bob S-
84, a native of Maineville.
died Friday. Jan.'2 at Blue.
Ash Nursing Home. _
He was a graduate engi-
neer of the University of
Cincinnati and was retired from Coulter
Electronics inc. of Hialeah, Fla. "
He was Past Patron of_ Fairrhount
Chapter 352 OES, Past Master of
Excelsior Lodge 369 F&AM. member of
the Price Hill Chapter 164 RAM.
McMillan Lodge 141 F&AM, Valley of
Cincinnati Ancient Accepted Scottish
Rite, and was a member of the Crystal
River. Fla. United Methodist Church.
He was precztled in death by his wife
of 53 years, Jean Blum Innis; parents
Lucian and Lulu Mae Innis; brothers
Howard L. and Charles F. Tad Innis:
sisters-in-law Bette B. Innis and Minarn
B. Innis; and brotherin-law Robert
McDaniel.
He is survived by son James R. Jr.
and daughter-in-law Carolyn C. of
Harrison; grandchildren Julia G. of
Baltimore, Md. and Robert P. of San
Francisco; sisters Mildred Holden and
husband Travis of Urbana and Janet
McDaniel of Lebanon: _brothers Lester
and wife Ruth B. of Montgomery and
John William Joe of Dayton; brother-
in-law Walter Blum Jr. and wife Virginia
S. of Maineville; and 18 nieces and
nephews.
Funeral service was held Tuesday.
Jan. 6 at Paul R. Young Funeral Home,
Mt. Healthy with visitation Monday, Jan.
5. ' -
Burial was at Baltimore Pike
Cemetery, Cincinnati. Fairmount 352
OES services were held Monday.
5. /RIEL /'17/5i ' i I
omknemn /ff? ~
Dick A. Irelan. 86. of
Waynesville died Saturday, Dec.
19 at OtterbeinLebanon.
He graduated from Steele High
School in Dayton and Ohio
University in~_Athens. where he
was a Beta Theta Pi.
He was a member of the First
Church of Christ Scientist.
Centerville. Sons of the American
Revolution Montgomery Chapter,
Dayton, Dayton Masonic Lodge
147 F& AM, Scottish Rite, York
Rite and Antioch temple all in
Dayton, OES Chapterl07 in
Waynesville, the Warren County
Shrine Club, the Little Miami
River Association charter mem-
ber! and past president and grand
marshal of the Warren County
Fair Board.
He was preceded in death by
his sister Wilma Knowlton. .
He is survived by his wife Luiie
,§M.; four daughters and sons-in-
"'law Patricia A._ and Mark Mason
of Waynesville, Lynne M. and
Kent Lindsey of Chicago, Deborah
J. and Gary Harlow of
Beavercreek and Mary Beth Irelan
of Georgetown, Ky.; grandchildren
Victoria Williams, Jennifer
Miroballi, Vance Va_ir,- Gregg
Harlow. Jeff Lindsey, Grant
Harlow, Tiffany'Wi1s0n-Mobley.
Vanessa Woods, Hailey Beth
Wilson and Layne Peden: nine
great-grandchildren; and brothers
Frank of Texas and Patrick of
Waynesville.
Funeral service was Tuesday,
Dec. 22 at StubbsConner
Funeral Home, Waynesville.
If desired, contributions may
'be made to Shrlners Hospitals or
the First Church of Christ
Scientist, Centerville.I54/
- 92
Clayton lvms /f? 59 W
Clayton Blake Ivins. 85, of
Lebanon died Saturday. Dec. 5.
He was born Nov. l. 1913 in
Tulahoma, Tenn. to Harry L. and
Mabel Blake! Ivi_ns. He was a
World War ll U.S. Army veteran.
He worked for both Warren
County and Turtlecreek Township
as a mechanic before retiring from
Doebler Brothers Inc. in 1978. I-Ie
was a member of the Bethany
United Church of Christ.
He-was preceded in death by
his parents; brothers Charles,
Leroy, Edward and Robert; and
sisters Dorothy, Grace and June.
Survivors include his.w'ife of 52
years. Shirley St. John! Ivins of
Lebanon; sons Richard of
Cincinnati and Randy and his wife
Cyndi of Evendale; daughter Gail
and her husband J eff Clark of
Cincinnati; sisters Jean Dunn and
Catherine Jones both of Lebanon,
Esther Carey of Kissimmee. Fla.
and Helen_Hauck of Hamden,
Ohio; a'ntl"g"andchildren Rachael
and Nicholas -Ivins.
Visitation was Tuesday, Dec. 8
with funeral service Wednesday,Dec. 9_ all at Oswald-Hoskins
Funeral _H<>'mé. Lebanon with
Pastor Allen Wentworth oiciating.
Interment was at Bethany
Cemetery. _
- Memorials are requested to the
Alzheimer's Association, 644 Linn
st, Suite 1026, Cincinnati, Ohio
45203. ' -8
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1 The followin investigation was conducted by Special* Agent @n O6/28/2°°5=
. Writer accessed www.uspto.gov, operated by the United
States Department of Commerce, Patent and Trademark Office.
Using the search engine at www.uspto.gov, a search was conducted
for all granted patents and applications that listed "Ivins" as
an inventor. The database searched included all patents since
1976. The search engine revealed two patents listing Ivins,
Bruce on the inventor line. These patents are as follows:
1! ."Asn0ro¢renic B anthracis exmesao yF |1vins9292Bruce_ Frederick, MDT. The patent was
originafly filed on November 23, 1994 and was granted on November
13, 2001 under the patent number 6,316,006.
2! "Method of making a vaccine for anthrax," invented b Ivins;Bruce Frederick; MDLJ K
H Fie patenéwas originally filed on March 7, 2000 and was granted on May 14,
2002 under the patent number 6,387,665.
A search was also conducted for[::::::::::]in the above
mentioned website. The only result returned from this search was
the patent labe1ed| Imentioned above. Based on this search, it appears as if| |
only has one patent under his name.
Printouts from the abovementioned searches for each of
the patents are attached to and made part of this document.
Administrative: The abovementioned search was
performed in response to emails found during a review of
electronic evidence gathered during the investigation into Major
Case 184. The emails in question were authored by BRUCE IVINS on
April 15 and 16, 1999. The emails discussed the process involved
with filing the patent. Copies of these emails are attached to
and made part of this document.
1
1
92
United States Patent: 6,387,665 Q ' Page 1 of 11
.
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United States Patent 6,387,665
Ivins , et al. May 14, 2002
Method of making a vaccine for anthrax
Abstract
A method of making a vaccine for anthracis that inolves a bacterial expression system and production
and use of protective antigen PA! against Bacillus anthracis. The PA immunogen is useful in a vaccine
against human anthrax. The PA can be produced by an asporogenic organism which produces the
desired antigen, which is then harvested from the supematant.
Inventors: Ivins; Bruce Frederick, MD!; Worsham; Patricia Jefferson, MD!; Friedlander; Arthur
M. Gaithersburg, MD!; Farchaus; Joseph W. Frederick, MD!; Welkos; Susan L.
Frederick, MD!
Assignee: The United States of America as represented by the Secretary of the Army
Washington, DC!
App1.No.: 520215
Filed: March 7, 2000
Current U.S. Class: 435/71.1; 424/ 1 84.1; 424/234.1; 424/246.1; 435/69.1;
435/69.4; 435/252.3; 435/252.31; 435/320.1; 435/485;
_ 530/3 50
Intern'l Class: C12P 021/04
Field of Search: 424/ 1 84.1,234.1,246.1 530/350
435/69.1,69,71.1,320.1,172.1,172.3,252.3,252.31,200.1
References Cited [Referenced By}
U.S. Patent Documents
3208909
4455142
5071748
5077214
http://patft.uspto.gov/netacgi/nph-Parser?Sectl=PTO2&Sect2=I-IITOFF&u=/netahtrnl/sear... 6/28/2005Sep., 1965
Jun., 1984
Dec., 1991
Dec., 1991Puziss et al.
Martins et al.
Miller. -
Guarino et al.
United States Patent: 6,387,665 . . Page 2 of 11
L
5081029 Jan., 1992 Zarling et al.
Other References
Ivins et al. European J. Epidemiology. Mar. 1988. 4!: 12-19*
Ivins et al. Clin. Immunology Newsletter. 9!: 30-32, l988.*
Uptake of Congo red by Virulent Strains of Bacillus Anthracis, Worsham et al., 1991 ASM
Abstracts, p. 75.
Understanding Biotechnology Law, edited by Gale R Peterson, Marcel Dekker, Inc., date
unknown!.
Vaccine Efcacy of Bacillus anthracis Protective Antigen Produced in Pokaryotic and
Eukaryotic Cells, Ivins, et al., Inst. Infect. Dis., Ft. Detric Frederick, MD, May 25, 1994.
Immunization Against Anthrax with Aromatic Compound Dependent Aro-! Mutants of
Bacillus anthracis and with Recombinant Strains of Bacillus subtilis that Produce Anthrax
Protective Antigen; Ivins, et al., Infect. and Immunity, Nov. 1986, vol. 54, No. 2, pp. 537-
542.
Cloning and Expression of teh Bacillus anthracis, Ivins, et al.; Infect and Immunity, May
1986, vol. 52, No. 2, pp. 454-458.
Expression of the Bacillus antracis Protective Antigen Gene by Baculovirus and Vaccinia
Virus Recombinants; Icono-Connors, et al., Infect. and Immunity, Feb. 1990, vol. 5 8, No. 2,
pp. 366-3 72.
Protection against Anthrax with Recombinant Virus-Expressed Protective Antigen in
Experimental Animals; Iacono-Cormors, et al., Infect. and Immunity, Jun. 1991, vol. 59, N0.
6, pp. 1961-1965.
Primary Examiner: Graser; Jennifer E.
Attorney, Agent or Firm: Arwine; Elizabeth, Moran; John Francis, Harris; Charles H.
Parent Case Text
This application is a divisional application of Ser. No. 08/346,238 led Nov. 23, 1994, said application
allowed.
Claims
What is claimed is:
1. A method of making a vaccine comprising: incorporating a protective antigen produced by
recombinant asporogenic B. anthracis with a pharmaceutically acceptable carrier, wherein said
recombinant asporogenic B. anthracis was isolated from a .DELTA.Sterne-1 pPA102! strain of bacteria
and said recombinant asporogenic B. anthracis does not have the ability to bind a dye when grown on
Congo Red Agar.
2. The method of claim 1, wherein the recombinant asporogenic B. anthracis is B.
Anthracis .DELTA. Steme-1 pPA 1 02! CR4.
http://patftuspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=I-IITOFF&u=/netahtml/sear... 6/28/2005
r
United States Patent: 6,387,665 . _ Page 3 of 11
v
92
3. The method of claim 1, wherein the vaccine is in the form of a suspension.
4. The method of claim l wherein the vaccine is in the form of buffered suspension.
5. The method of claim 1 wherein said carrier is an adjuvant.
Description
FIELD OF THE INVENTION
This invention relates to the bacterial expression system, production and use of protective antigen PA!
against Bacillus anthracis. The PA immunogen is useful in vaccine against human anthrax. The PA can
be produced by an asporogenic organism which overproduces the desired antigen, which is then
harvested from the supernatant. _
BACKGROUND OF THE INVENTION
Bacillus anthracis is the etiologic agent responsible for anthrax, a disease often found in persons
exposed to infected animals or their products. Persons particularly exposed to animals include
veterinarians, laboratory technicians, ranchers and employees working with skin or hair of animals. The
mode of entry into the body may be the skin or, when contaminated meat is eaten, the gastrointestinal
tract. Inhaling of spores can causeinhalation anthrax, a disease that can be fatal. Vaccines against
Bacillus anthracis have been available. Vimlent strains of the organism produce two toxins and a poly-
D-glutamic acid capsule which are coded for on two endogenous plasmids, pX0l and pX02,
respectively. Loss of either of the plasmids results in an attenuated strain of reduced virulence, while
loss of both results in an avirulent organism. The history of the USAIVIRIID Sterne strain of B. anthracis
prior to 1981 is uncertain, though it is believed to be derived from the Steme strain isolated at the
Onderstpoort Research Laboratory in Pretoria, South Africa.
In 1985 the Bacillus anthracis protective antigen PA! gene was cloned into a plasmid pUB1 10!
resulting in the formation of a recombinant plasmid identied as pPA102, which was reported in the
literature Ivins and Welkos, Infection and Immunity, 54:537-542 986!!. The production of vaccines
lacking lethal factor was possible thereby. However, a primary problem remained, since the Bacillus
anthracis formed spores. Once spores have formed, they persist in the environment for months and
years. Once the laboratory environment contains such spores, it is very difficult to free the environment
of the spores.
It was also previously reported that protective antigen PA! could be produced in baculovirus. [Iacono-
Connors, et al., Infection and Immunity, 58:366-372 990!; Iacono-Connors, et al., Infection and
Immunity, 59:1961-1965 99 1!] A major problem in production of the PA in the baculovirus disclosed
therein is that the desired antigen requires a complex purication process. Even aer purication by
irnmuno-aflinity chromatography, undesired cellular material continues to contaminate the desired
product.
DETAILED DESCRIPTION OF THE INVENTION
The instant invention provides organisms which produce protective antigen PA! lacking lethal factor
and edema factor proteins which, when present as contaminants in vaccine, can cause serious side '
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eects. The producing organisms of the invention are also, surprisingly, non-sporulating. Furtherrnore,
the desired antigen is expressed into the supernatant. Hence, the protective antigen produced is easily
puried and, though protective, does not cause many of the troublesome side effects of prior art
vaccines. The organisms of the invention lacking spore-forming function may be killed by heat shock at
temperatures as low as 60.degree. C. for 60 minutes. Hence, contamination of the environment with
viable spore-forming organisms is easily avoided and decontamination is easily accomplished.
Genesis of .DELTA.Sterne-l pPAl 02!CR4:
A 6 kb Bam HI fragment harboring the PA structural gene isolated from the endogenous Sterne plasmid
pXO1 was ligated into plasmid pBR322 and cloned into Escherichia coli bacteria V odkin and Leppla,
1983!. From the resultant recombinant plasmid pSE36, the 6 kb agment was then subcloned into the
gram-positive vector pUB110 using the Bani H1 restriction site. The resulting plasmid was transformed
into B. subtilis IS53 and two stable PA producing, kanamycin resistant isolates were found pPA101 and
pPA102! Ivins and Welkos, 1986!. Subsequent analysis of the plasmids revealed that both had suffered
spontaneous deletions. The pPAl02 was found to have lost 4.2 kb of DNA from 363 bp 3' of the
kanamycin resistance gene to approximately 164 bp 5' of the start of the PA structural gene, a result
consistent with the observed inactivation of the phleornycin resistance gene of pUB1 10. The plasmid
was then electrotransformed into .DELTA.Steme-1, a plasmid-ee strain of B. anthracis Infection and
Immunity, 52:454-458 986! and transformants were selected for kanamycin resistance. Transformants
displaying a stable PA+, kanamycin resistant, LF-, EF-, capsule-! phenotype were selected. This strain,
.DELTA.Sterne-1 pPA102!, was then subjected to Congo Red agar selection for mutants displaying an
inability to bind the dye, a characteristic known to correlate with an asporogenic phenotype W orsham,
submitted!. The selected isolate, now designated .DELTA.Steme-1 pPA102!CR4 was further
subcultured three times to insure that a single clone was isolated. This clone has served as the seed stock
for all research and development of fermentation conditions, and purication of PA.
Materials and Methods:
Fermentation Conditions
Media: FA medium was used for all plates and liquid cultures described here unless otherwise specied.
FA medium consisted of 33 g/1 tryptone Difco!, 20 g/l yeast extract Difco!, 2 g/l L-histidine, 8 g/l
Na2HPO4, 7.4 g/l NaCl, 4 g/1 KH2PO4 adjusted to pH 7.4 with NaOH.
Precultures: A working stock of .DELTA.Sterne-1 pPA102!CR4 was prepared from the seed culture by
streaking cells on an FA medium plate containing 40 .mu.g/ml of kanamycin. A sweep from the
conuent growth zone on plate was cultured one time in liquid FA medium supplemented with
kanamycin 40 .mu.g/ml to a nal O.D..sub.600mn of 4.0. This culture was checked for purity by
streaking on SBA plates, and diluted into multiple vials containing sterile 100% glycerol to a nal
glycerol concentration of 50% v/v!. These stocks were stored at -70.degree. C. A single vial was
removed at the start of each fermentation cycle and discarded after use. The defrosted cells were
streaked onto FA plates containing 40 .mu.g/ml kanamycin and incubated at least 16 hrs at 37 .degree. C.
After 16 hrs the plated cells were used to inoculate 50 mls of FA medium supplemented with
40 .mu.g/ml kanamycin in a 250 ml baffled-Erlenmeyer ask Bellco Laboratories!. The culture was
incubated at 37.degree. C. at 200 rpm for 6 hrs or until an O.D..sub.600nm of 4-6 was obtained. The
cells were then subcultured into 50 mls of FA medium in an identical ask under identical conditions.
After 6 hrs, or a culture O.D..sub.600mn of 6.2-6.5, a 1.6% v/v! inoculum was transferred to 300 mls of
PA medium supplemented with 40 .mu.g/ml kanamycin in a 2 liter bafed Erlemneyer and incubated at
37.degree. C. at 200 rpm for 7 hrs, or until a nal O.D..sub.600nm of 3.5-3.7 was achieved.
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Fermentation conditions: The fermentations described here were carried out using a New Brunswick
Bio-Flo 3000 equipped with a 5.0 liter working volume glass vessel and stainless steel headplate and
hemispherical bottom cooling dish. Four liters of FA medium were added to the vessel, which had been
previously completely disassembled, scrubbed in a dilute Envirochem solution and autoclaved for 15
min after the addition of 4 liters of H20. The polarographic DO.sub.2 probe Ingold! and pH probes
either liquid or gel lled, Ingold! were also inserted and all addition and sampling ports were sealed or
clamped and wrapped in aluminum foil. Addition lines consisted of surgical grade autoclavable Tygon
tubing Thomas Scientic! and all lines were sealed with the exception of the condenser, which was left
open to permit pressure release, but covered with aluminum foil. The vessel was autoclaved using a 10
min exposure time at 121 .degree. C. and removed from the autoclave as soon as sufficient cooling had
occurred to allow opening of the autoclave. The vessel was then immediately connected to the fermentor
unit and the condenser line was connected to a sterile liquid trap and 0.2.mu. capsule lter to avoid the
introduction of contaminants during the cooling process. The vessel was then cooled to 37.degree. C.
using the fermentor driven temperature control and positive pressure was provided using compressed
sterile ltered air. Once the vessel had cooled to 37.degree. C. sterile ltered kanamycin was added to a
nal concentration of 40 .mu.g/ml. The agitation was activated at 150 rpm and aeration was adjusted to
l-1.2 volume/volume/min vvm! and antifoam C DOW!, that had been diluted 10-fold into H.sub.2 O
and autoclaved, was added to a nal concentration of 200 ppm.
A preinoculation sterility check was conducted for a minimum of 16 hrs during which time pH, agitation
and temperature were continually monitored. After the 16 hrs required for DO.sub.2 probe polarization,
the DO.sub.2 was also monitored along with turbidity. The D.sub.2 probe was calibrated using an
INGOLD calibration device which sets the zero value to 4 mA and 100% to the oxygen tension
determined by the solubility of oxygen in the medium after aeration and agitation at 37.degree. C. The
calibration and response of the electrode was then checked by sparging with pure N.sub.2. The vessel
was judged to be sterile if the pH and DO.sub.2 remained constant and no increase in turbidity was
observed. It should be emphasized that the short autoclave cycle for vessel sterilization was required to
minimize caramelization, Millard and other chemical degradation reactions which are problematic due
to the high concentrations of yeast extract and tryptone in FA medium. As an additional conrmation of
sterility, 50 mls was aseptically removed from the fermentor to a 250 mls Erlenmeyer and incubated at
37.degree. C. at 200 rpm for 48 hrs with no sign of growth. Under the conditions outlined here
contamination has not been observed in more than 10 fermentation cycles. .
Once the sterility of the vessel had been veried, the 300 ml inoculum described above was added to the
vessel through the addition port of the headplate and the initial O.D..sub.600nm was recorded. A sample
of the inoculum was also streaked on SBA plates and incubated for 48 hrs at 37.degree. C. to verify .
inoculum purity. Using the Bio-Flo 3000, aeration was maintained at 75% of saturation by increasing
agitation from the initial 150 rpm to a maximum of 400 rpm and ultimately by supplementing the 1 vvm
aeration rate with pure oxygen. The mixture rate and percentages of air and oxygen were controlled by a
solenoid and algorithm developed by Nev Brunswick Scientic. Both gases had a working pressure of
approximately 10 psi.
The O.D..sub.600nm dry cell weight DCW!, production of PA, DO.sub.2, pH, agitation and
temperature were monitored throughout each fermentation cycle. The O.D..sub.600nm DCW and PA
production analysis were carried out by manually sampling the fermentation liquor at hourly intervals
using a sterile sampling port. O.D..sub.600nm was measured after dilution of the culture using sterile
medium prepared for that fermentation. For each O.D.600 determination, two appropriate dilutions were
made and results were considered acceptable only when both dilutions yielded a linear response. DCWs
were determined starting with a 2 hr point by centrifuging 10 mls of fermentation liquor at
1l,953.times.g for 10 min, resuspending the cell pellet in 10 mls of sterile PBS and pelleting the cells
again under the same conditions. The cell pellet was resuspended in a minimal volume of PBS and
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transferred quantitatively to a preweighted Eppendorf centrifuge tube and centrifuged at 14,000 rpm for
5 min. Excess PBS was removed and the cell pellet was dried in a speed-vac for 72 hrs under vacuum
and a medium heat setting. A nal analysis of the dry weight versus O.D..sub.600mn revealed that the
relationship between the two parameters was adequately t with a linear function.
Fermentation Reproducibility: The reproducibility of the cell growth parameters, biomass and PA
production in fermentations carried out with the Bio-Flo 3000 under the conditions described above
have been summarized in Table I below. Two fermentations were carried out at 75% of the maximum
dissolved oxygen concentration in a strict batch mode with no pH control or additions other than
antifoam C. The variation in the agitation rate during the rst 100 min of the fermentation was the result
of the AGDO.sub.2 agitation DO.sub.2! control mode chosen to maintain the dissolved oxygen tension
at 75 % of the maximum. Briey, this algorithm attempts to control the oxygen tension by rst altering
the agitation rate until this proves insufcient, at which point the process air is supplemented with pure
oxygen as needed to maintain the desired DO.sub.2. The temperature was held constant at 37 .degree.+/-
0.1.degree. C. The pH was monitored, but not regulated as an internal check on the aeration of the vessel
during the course of the fermentation. The fact that the pH revealed a decrease on only 0.2 pH units in
the rst 150 min was consistent with an aerobic culture metabolizing the limited carbohydrate supplied
with the yeast extract to CO.sub.2 and organic acids. Once the carbohydrate was exhausted after ca. 150
min, the bacillus switched to the utilization of amino acids and peptides for a carbon source, which
under aerobic conditions resulted in ie release of NH.sub.4 OH and the observed increased culture pH.
These fermentations were sampled on an hourly basis and allowed to proceed until no fL1I.'|IhCl increase in
O.D..sub.600nm was observed over two time points. O.D..sub.600nm, DCW analysis and product
measurements were carried out for each sample as described above. Samples for PA production were
sterile ltered followed by the addition of HEPES and the complete protease cocktail as described under
PA quantitation. The samples were concentrated, desalted and ultimately concentrated 80-fold prior to
being analyzed using SDS-PAGE. The major band of the gel corresponded to the 83 kDa PA product.
An increasing in the intensity of the protein band was seen with increasing fermentation time. Study of a
Westem blot of another time course of a batch fermentation was developed with polyclonal rabbit anti-
PA83. Comparison revealed that along with increasing PA 83 kDa there was also a pronounced increase
in the abundance and form of proteolytic degradation products of PA.
TABLE 1
Summary of Aerobic .DELTA.Sternel pPA102!CR4 Fermentations
Final
Final Final Yield Doubling
Conc. Yield mg Specific Time
Fermentation .mu.g PA83/ mg PA83/g Growth T.sub.D
Conditions ml PA83! DCW! Rate min!
Aerobic, Batch 51 235 8.10 0.0132 min.sup.l 53
Aerobic, Batch 64 301 10.7 0.0136 min.sup.~1 51
Aerobic, Batch 45 225 7.40 0.0136 min.sup.l 51
pH constant
Aerobic, 68 360 ND 0.0116 min.sup.l 60
FedBatch
non-
continuous!
DCW = dry cell weight
The data presented in Table 1 demonstrated that the PA yield on a unit volume and biomass basis, as
well as the cell growth parameters, were reproducible for the batch fermentations conducted without pH
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control. The nal fermentation pH values of 8.57 and 8.67 after an elapsed fermentation time of ca. 8 hrs
were also comparable. The effect of prolonged exposure to these mildly alkaline conditions on cell
growth, PA production and subsequent degradation was investigated by repeating the fermentation at a
constant pH of 7.50+/-0.05 pH units. This was accomplished using the immersed vessel pH probe and
automated additions of 2 N HCI or 1 N NaOH. The results shown in Table 1 demonstrate that there was
no clear effect of constant pH on any of the parameters evaluated. SDS-PAGE analysis of the
fermentation time points sampled for PA production also revealed no signicant differences.
The nal fermentation presented in Table 1 was a noncontinuous fed-batch trial during which 1/10
volume of a 10-fold concentrate of sterile-ltered tryptone was added after 5 hrs or an O.D..sub.600mn
of 7.5. The result suggested that such fedbatch fermentations provide possible protocols for
improvement to increase yield and decrease proteolysis.
Harvest conditions: Fermentations were allowed to proceed until no further increase in O.D..sub.600mn
was observed. At this point, the fermentor was cooled to 10.degree. C. and the protease inhibitors
phenylmethylsulfonyl uoride PMSF!, 1,10-phenanthroline OP! and ethylenediamine tetraacetate
EDTA! were added to nal concentrations of 0.1, 0.05 and 2 mM, respectively. The cells were then
pumped from the fermentor vessel at room temperature using an Amicon DC10L concentrator equipped
with a 10-:.sup.2 0.1 .mu.polysulfone hollow-ber cartridge. The fermentor liquor was diluted 1:1 with
25 mM diethanolamine DEA!, 50 mM NaCl, 2 mM EDTA, 0.1 mM PMSF adjusted to pH 8.9 with
HCI. The ltrate was collected at an operating pressure of less than 20 psi and transferred directly to a
second Amicon DCIOL equipped with two 30 kDa cutoff 10-ft.sup.2 wound spiral cellulosic cartridges.
The ltrate was concentrated approximately 10-fold before being subjected to dialtration at an
operating pressure of less than 30 psi against the same buffer. The conductivity of the retentate was
monitored with an Amber Sciences conductivity meter and platinum immersion pencil-type electrode.
The dialtration step generally required 20 liters of buffer, but was considered complete only after the
conductivity of the concentrated retentate was equivalent to that of the starting buffer.
Quantitation of 83 kDa PA in crude fermentation liquor: The fermentation liquor was sampled using a
sterile port at regular intervals throughout the fermentation process. The samples for PA determination
were ltered through syringe type 0.2.mu. cellulose acetate lters, 0.1 mM PMSF, 2 mM EDTA,
50 .mu.M OP and 20 mM HEPES pH7.3 were added and the samples were frozen at -70.degree. Cl The
samples were defrosted on ice and concentrated using Amicon Centricon 30 concentrators at
45 00.times. g. The samples were concentrated approximately 10-fold, diluted to the original volume with
10 mM TRIS pH8.0, 0.1 mM PMSF, 2 mM EDTA, 0.05 pM OP and concentrated again. The
concentrated sample was desalted again using the same buffer, frozen and nally lyophilized using a
Speed-Vac. The dried samples were dissolved in 25 .mu.l of the TRIS buffer described above and
diluted 1:1 with a 2.times.SDS solubilization buffer consisting of 50 IIM Na.sub.2 CO.sub.3, 4% w/v!
SDS, 12% v/v! glycerol, 2% v/v! 2-mercaptoethanol and 0.01% W/v! Bromphenol Blue prior to
heating at 95.degree. C. for 5 min. The fermentation samples containing varying amounts of PA 83 kDa
were solubilized as described above and run on a Daiichi 4-20% gradient TRIS/TRICINE gel to
approximate total yield of PA. Two hlmdred to 2000 ng samples of puried PA were solubilized in the
same buffer and loaded onto the gel in constant total volume of 3 .mu.1._ Three or four appropriate
dilutions of the fermentation samples determined from the rst gel were loaded onto the gel with the
standards and electrophoresed at 100 V initially and 140 V once the samples entered the separating gel
and until the Bromphenol Blue dye reached the bottom edge of the separating gel. The gel was then
xed in 10% v/v! acetic acid 20% v/v! MeOH for 10 min, rinsed with MQ H.sub.2 O and stained with
Coomassie Brilliant Blue 0.05% W/v! in 10% v/v! acetic acid for a minimum of 16 hrs to allow
complete and uniform staining. The stained gel was then destained in 10% v/v! acetic acid until the
background contained no visible residual dye. The gel was then scanned on a laser densitometer LKB,
Ultrascan XL Laser Densitometer!. Representative portions of the gel without protein were randomly
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chosen and scanned to determine backgrotmd absorption for an accurate baseline. The region to be
scanned for each lane containing PA was then visually aligned to insure that the entire protein peak and
adequate baseline were included in each scan. The scans were completed and the integration values were
determined using the LKB preprogrammed Gaussian algorithm and later were conrmed by cutting out
individual peaks and manually integrating based on peak weight. The resulting integration values were
plotted using Sigmaplot J andel!. Linear regression of the results revealed typical r values of 0.992-
0.996. The linear standard curve was then used to quantitate the amount of 83 kDa PA in the various
fermentation samples based on the same integration methods.
Purication: The exact volume and conductivity of the PA in DEA buffer was determined and solid KCl
was added to the solution to a nal concentration of 30 mM and conductivity of 10-11 mmhos/cm. The
PA was pumped with a peristaltic pump through a monoQ column prepared by collecting 100 mls of
hydrated Bio-Rad Macro Prep 50Q on a sintered glass lter and washing sequentially with 1 liter of 25
mM DEA, 50 mM NaCl, 1 mM EDTA, 50 .mu.M OP and 0.1 mM PMSF pH8.9 and 1 liter of the same
buffer with 30 mM KCl added. The conductivity 0-11 mmhos/cm! and pH of 8.9 of the eluate om
the Macro Prep 50Q after the second wash were comparable to that of the PA solution after addition of
KCI. The Macro Prep 50Q resin was then degassed and slurry packed into a Pharmacia K column with a
Rainin Rabbit-Plus peristaltic pump at 48 rpm and a ow rate of 15 mls/min. The nal column volume
was .times.5 cm! 98 mls. The PA solution was pumped through the Macro Prep 50Q column at a rate
of 10 mls/min and the eluate was collected until all of the PA sample volume was loaded and the column
washed with an additional 100 mls of DEA/KCI buffer. The eluate containing unbound PA was
concentrated and dialtered using an 1-ft.sup.2 30 kDa cutoff cellulosic Amicon wound spiral cartridge
at an operating pressure of 20 psi.
The nal concentrate ca. 400 mls, 6-7 mmhos/cm! was passed through a 0.2.mu. cellulose acetate lter.
The ltered PA was loaded onto a Poros IIQ perfusion chromatography column using a quaternary
Waters 600E HPLC pump. The column was prepared by hydrating seven grams of the Poros IIQ
perision resin in twice the packed bed volume of 2% w/v! NaCl. After settling the resin was
resuspended in six times the packed bed volume of 25 mM DEA pH 8.9, 50 mM NaCl, 7.5% v/v!
ethylene glycol and allowed to settle overnight at room temperature. The resin was then resuspended in
three times the packed bed volume and fmally in one and one-half times the fmal volume before the
slurry was extensively degassed using a vacuum pump vacuum unknown!. The entire degassed slurry
was then transferred to a Waters AP 20.times.1O0 mm glass HPLC column and the column was packed
in one step using the Waters 600E pumps at a ow rate of 20 mls/min and a backpressure of 650 psi at
room temperature. The column separation efficiency was then tested at a flow rate of 10 mls/min using a
linear 1 M NaCl gradient and ovalbumin 5 mg/ml Sigma! and bovine serum albumin 10 mg/ml Sigma!
in DEA as buffer as standard proteins. Approximately 100 mls of PA ca. 20-30 mg PA! cooled to 4-
6.degree. C. was applied to the column and followed with a 20 min wash in the starting buffer at room
temperature to elute tmbound material. The column was then developed with a linear gradient to 30% of
the 1 M NaCl DEA elution buffer. The puried PA was found to elute between 10-15%, while the
smaller molecular weight proteolytic breakdown products eluted as a shoulder or partially resolved peak
at 16-20% of the elution buffer. The resolution of the two peaks was found to be a frmction of content of
PA proteolytic degradation products. The eluant was monitored at 280 nm and peak fractions were
collected by manual triggering of an ISCO fraction collector. Samples of the peak fractions were diluted
into 5-10 volumes of TRIS pH8.0, 0.l mM PMSF, 50 AM OP, 1 mM EDTA buffer and concentrated .
using Amicon Centricon 30 concentrators at 45 00.tirnes.g at 4.degree. C. to approximately the initial
sample volume. An equal volume of SDS-PAGE solubilization buffer was added to the sample
immediately prior to heating at 95 .degree. C. for 5 min. Purity was assessed from 8-25% SDS-PAGE
PHAST gels Pharmaoia! and actions with the highest purity were combined and dialyzed against 40-
50 volumes of 25 mM DEA pH8.9, 50 mM NaCl, 0.1 mM PMSF and 2 mM EDTA at 4.degree. C. for at
least 16 hrs. Fractions judged empirically to be less than 95% pure were rechromatographed under the
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same conditions and purity of the fractions was reassessed as described above. All actions of greater
than 95% purity were ultimately combined, aliquoted and frozen at -70.degree. C. subsequent to
determination of the total PA concentration.
Analysis and characterization of purified 83 kDa PA: Puried PA was quantitated by measuring UV-
absorption at 280 nrn using the relationship of 1 A.sub.280nm in a 1 cm pathlength cuvette is equals 1
mg PA/ml Leppla, 1988!. Results obtained in this manner were conrmed using the Bio-Rad Bradford
protein assay under conditions suggested by the manufacturer. PA purity was assessed using SDS-
PAGE under conditions described above. Capillary electrophoresis analytical assays have also proven
promising in the assessment of PA purity and amounts of residual protease inhibitors in nal product.
Feasibility studies using a 47 cm.times.50 pm uncoated silica capillary and borate/SDS/acetonitrile
buffer revealed an excellent separation of the protein om residual protease inhibitors. Quantitation of
both protein and inhibitors has also proven possible, but the technique remains limited by the relatively
high limits of detection mM EDTA, 0.1 mM PMSF, and 0.05 mM OP! under current conditions.
Automated N-terminal sequencing was carried out with puried PA using an Applied Biosystems 470A
sequenator after desalting over Bio-Rad PDl 0 columns equilibrated with 5 mM NaC1 and 1 mM
CaCl.sub.2. A unique N~terminal sequence was found and the rst six residues of the sequence were
identical to PA from the endogenous plasmid pXO1 harbored by the USAMRHD B. anthracis Sterne
strain. In addition, the sequence corresponded exactly with the published DNA derived protein sequence
W elkos et al.!. Native gel electrophoresis under non-denaturing conditions revealed that PA puried
om .DELTA.Steme-l pPAl02!CR4 also exhibited the microheterogeneity noted previously for PA
produced by the Sterne strain. Cytotoxicity assays of the product using the macrophage lysis assay
F riedlander et al.! revealed that the titration curve of biological activity for PA from .DELTA.Steme-1
pPA102!CR4 was indistinguishable from that generated for PA from the Sterne strain.
Evaluation of .DELTA. Steme-1 pPA1 02!CR4:
EXAMPLE 1
B. Anthracis .DELTA.Sterne-l pPA102!CR4 was compared with its parent spore-forming strain B.
anthracis .DELTA.Sterne-1 pPA102!. Both organisms were plated onto sheep blood agar a preferred
medium for promoting bacterial spore production! and grown at 37 .degree. C. for 1 day, after which the
temperature was lowered to 25.degree. C. for 4 days. The two strains were also grown in liquid
Leighton-Doi medium, which is designed to promote spore production, for 1 day at 37.degree. C.
followed by 4 days growth at 25.degree. C. Growth om both agar and broth cultures were examined
under phase contrast microscopy for the presence of spores. Growth om all four cultures were then
resuspendedin phosphate buffered saline to a concentration of about 10.sup.9 colony-forming units
CFU! per ml. All four cultures were then heat shocked at 64.degree. C. for 60 minutes to kill vegetative
cells. Aliquots of 0.1 ml of the heat shocked material was then plated out onto sheep blood agar and
incubated at 37.degree. C. for 2 days.
Results:
B. anthracis .DELTA.Sterne-1 pPA102!: Spores were seen under microscopic examination of material
from both the sheep blood agar cultures and the Leighton-Doi medium cultures. On sheep blood agar
plates containing heat shocked culture material from both sheep blood agar cultures and Leighton-Doi
medium cultures, there was conuent growth. The data clearly indicate that B.
anthracis .DELTA.Sterne-1 pPA102! forms spores.
B. anthracis .DELTA.Sterne-1 pPA102! CR4: No spores were seen under microscopic examination of
material from both the sheep blood agar cultures and the Leighton-Doi medium cultures. On sheep
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blood agar plates containing heat shocked cultures, there was no growth whatsoever. The data clearly
indicate the B. anthracis .DELTA.Steme-1 PPA102! CR4, which has been deposited in the American
Type Culture Collection and has been assigned ATCC designation 69714, does not form spores. The
deposit at the American Type Culture Collection located at 12301 Parklawn Drive, Rockville, Md.
20852, USA was made on Nov. 16, 1994.
EXAMPLE 2
B. anthracis .DELTA.Sterne-1 pPA1 02!CR4 was grown in an FA medium fermentor culture. No spores
were seen upon phase contract microscopic examination. Only medium~length and long chains of bacilli
were seen. Dilution plate counts on the culture determined that the culture contained 1.86.times.l0.sup.9
CFU per ml. Three ml of culture was heat shocked at 60.degree. C. for 60 minutes, then 0.2 ml was
plated onto each of 5 plates of Tryptic soy agar. Aer incubation for 2 days at 37 .degree. C., no colonies
were seen on the agar plates, indicating that spore production in the fermentor was less than 1 per
1.86.times.10.sup.9 CFU. On two other fermentation nms with this strain, similar results were obtained.
No revertants to the parent spore-forming phenotype were observed.
The above process using an FA medium fermentor culture was repeated using the parent strain B.
anthracis .DELTA.Sterne-1 pPA102!. Growth on the tryptic soy agar after heat shock resulted in a total
of 1000 total colonies, indicating that the parent strain B. anthracis .DELTA.Sterne-1 pPA102! had
about 1000 spores per ml in the FA medium, or 1 spore per 106 CF U in the non-heat shocked medium.
EXAMPLE 3
Protective antigen PA! was prepared in accord with the teachings under Materials and Methods as
described above. The pmied. PA of B. anthracis .DELTA.Stern-1 pPA102!CR4 was mixed in diierent
buffers phosphate buffered saline, HEPES, Tris, glycyl glycine GG!, sodium citrate, for example! and
combined with monophosphoryl lipid A MPL!, Squalene, Tween 80 and lecithin. The mixture was then
lyophilized. At O and 4 weeks, vials of lyophilized MPL/PA/emulsion were reconstituted in phosphate
buffered saline PBS! and injected in 0.5 ml doses containing 50 .mu.g of PA per dose. At 10 weeks, the
guinea pigs were aerosol challenged with approximately 36 medial lethal doses of virulent Bacillus
anthracis spores of the Ames strain. The following data shows status two Weeks after the challenge.
Vaccine S/T* % Anti~PA**
PA in PBS + MPL emulsion! 10/12 83 29,427
PA in GG + MPL emulsion! 14/16 88 23,713
PA in Tris + MPL emulsion! 15/16 94 27,384
PA in HEPES + MPL emulsion! 15/15 100 25,482
PA in Citrate + MPL emulsion! 16/16 100 31,622
PBS 0/4 O <10
*Survived/Total, day 14 postchallenge
**Prechallenge serum titers to PA were determined by enzyme linked
immunosorbent assay. The geometric mean reciprocal titers were calculated
for each group and are expressed in this table.
*=t*~**
Una es
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United States Patent 6,316,006
Worsham , et al. November 13, 2001
Asporogenic B anthracis expression system
Abstract
. This invention relates to a bacterial expression system for production of protective antigen PA! against
bacillus anthracis. Recombinant asporogenic B. anthracits that are derived from .DELTA.Steme-1
pPA102! and show inability to bind the dye when grown on Congo Red Agar can be screened and
asporogenic strains isolated using methods of the invention. organisms of the invention lacking spore-
forming function may be killed by heat shock at temperatures as low as 60.degree. C. for 60 minutes.
Hence, contamination of the enviromnent with viable spore-forming organisms is easily avoided and
decontamination is easily accomplished.
Inventors: Worsham; Patricia Jefferson, MD!; Friedlander; Arthur M. Gaithersburg, MD!; Ivins;
Bruce Frederick, MD!
Assignee: The United States of America as represented by the Secretary of the Army
Washington, DC!
Appl. No.2 346238
Filed: November 23, 1994
Current U.S. Class: 424/246.1; 435/252.3; 435/252.31; 435/485
Intern! Class: C12N 015/00
Field of Search: 424/246.1 435/ 172.1,172.3,252.3,252.31,200.1
References Cited |Referenced By]
Other References
Ivins et al. Abstr. Gen. Meet. Am. Soc. Microbiol. 94 meet. 150, May 1994.*
Ivins et al. Infect. Immun. Feb. 1990. 58!: 303-3 08.*
Ivins et al. Infect. Immun. Nov. 1986. 54!: 537-542.*
Ivins et al. Infect. Immun. May 1986. 52!: 454-457.
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i.
United States Patent: 6,316,006 6 Page 2 0f10
Primary Examiner: Housel; James C.
Assistant Examiner: Shaver; Jennifer
Attorney, Agent or Firm: Han-is; Charles H., Moran; John Francis
Claims
What is claimed is:
1. A recombinant asporogenic B. anthracis isolated from .DELTA.Sterne-1 pPA102! which shows
inability to bind the dye when grown on Congo Red Agar.
2. A B. anthracis of claim 1 which is B. anthracis .DELTA.Sterne-1 pPA102!CR4.
3. A composition comprising the organism of claim 1 in a growth medium.
.4. A composition comprising the organism of claim 2 in a growth medium.
Description
FIELD OF THE INVENTION
This invention relates to the bacterial expression system, production and use of protective antigen PA!
against Bacillus anthracis. The PA immunogen is useful in vaccine against human anthrax. The PA can
be produced by an asporogenic organism which overproduces the desired antigen, which is then
harvested om the supematant.
BACKGROUND OF TI-IE INVENTION
Bacillus anthracis is the etiologic agent responsible for anthrax, a disease often found in persons '
exposed to infected animals or their products. Persons particularly exposed to animals include
veterinarians, laboratory technicians, ranchers and employees Working with skin or hair of animals. The
mode of entry into the body may be the skin or, when contaminated meat is eaten, the gastrointestinal
tract. Inhaling of spores can cause inhalation anthrax, a disease that can be fatal. Vaccines against
Bacillus anthracis have been available. Virulent strains of the organism produce two toxins and a poly~
D-glutamic acid capsule which are coded for on two endogenous plasmids, pX0l and pX02,
respectively. Loss of either of the plasmids results in an attenuated strain of reduced virulence, while
loss of both results in an avirulent organism. The history of the USAMRIID Sterne strain of B. anthracis
prior to 1981 is uncertain, though it is believed to be derived from the Sterne strain isolated at the
Onderstpoort Research Laboratory in Pretoria, South Africa.
In 1985 the Bacillus anthracis protective antigen PA! gene was cloned into a plasmid pUBl10!
resulting in the formation of a recombinant plasmid identied as pPAl 02, which was reported in the
literature Ivins and Welkos, Infection and Immunity, S54:537-542 986!!. The production of vaccines
lacking lethal factor was possible thereby. However, a primary problem remained, since the Bacillus
anthracis formed spores. Once spores have formed, they persist in the environment for months and
years. Once the laboratory environment contains such spores, it is very difcult to free the environment
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of the spores.
It was also previously reported that protective antigen PA! could be produced in baculovirus. [Iacono-
Connors, et al., Infection and Immunity, 58:366-372 990!; Iacono-Connors, et al., Infection and
Immunity, 59:l961-1965 991!] A major problem in production of the PA in the baculovirus disclosed
therein is that the desired antigen requires a complex purication process. Even after purication by
immuno-afnity chromatography, undesired cellular material continues to contaminate the desired
product.
DETAILED DESCRIPTION OF THE INVENTION
The instant invention provides organisms which produce protective antigen PA! lacking lethal factor
and edema factor proteins which, when present as contaminants in vaccine, can cause serious side
effects. The producing organisms of the invention are also, surprisingly, non-sporulating. Furthermore,
the desired antigen is expressed into the supernatant. Hence, the protective antigen produced is easily
puried and, though protective, does not cause many of the troublesome side effects of prior art
vaccines. The organisms of the invention lacking spore-forming function may be killed by heat shock at
temperatures as low as 60.degree. C. for 60 minutes. Hence, contamination of the environment with
viable spore-forming organisms is easily avoided and decontamination is easily accomplished.
Genesis of .DELTA.Steme-1 pPA102!CR4:
A 6 kb Bam I-II agment harboring the PA structural gene isolated om the endogenous Sterne plasmid
pX01 was ligated into plasmid pBR322 and cloned into Escherichia coli bacteria V odkin and Leppla,
1983!. From the resultant recombinant plasmid pSE3 6, the 6kb fragment was then subcloned into the
gram-positive vector PUB110 using the Bam H1 restriction site. The resulting plasmid was transformed
into B. subtilis IS53 and two stable PA producing, kanamycin resistant isolates were found pPAl0l and
pPAl 02! Ivins and Welkos, 1986!. Subsequent analysis of the plasmids revealed that both had suffered
spontaneous deletions. The pPAl 02 was found to have lost 4.2 kb of DNA from 363 bp 3' of the
kanamycin resistance gene to approximately 164 bp 5' of the start of the PA structural gene, a result
consistent with the observed inactivation of the phleomycin resistance gene of pUB1 10. The plasmid
was then electrotransformed into .DELTA.Steme-1, a plasmid-free strain of B. anthracis Infection and
Immunity, 522454-458 986! and transformants were selected for kanamycin resistance. Transformants
displaying a stable PA+, kanamycin resistant, LF-, EF-, capsule-! phenotype were selected. This strain,
.DELTA.Steme-l pPAl02!, was then subjected to Congo Red agar selection for mutants displaying an
inability to bind the dye, a characteristic known to correlate with an asporogenic phenotype W orsham,
submitted!. The selected isolate, now designated .DELTA.Steme-1 pPAl02!CR4 was further
subcultured three times to insure that a single clone was isolated. This clone has served as the seed stock
for all research and development of fermentation conditions, and purication of PA.
Materials and Methods:
Fermentation Conditions *
Media: FA medium was used for all plates and liquid cultures described here unless otherwise specied.
FA medium consisted of 33 g/l tryptone Difco!, 20 g/l yeast extract Difco!, 2 g/l L-histidine, 8 g/l
Na2I-IP04, 7.4 g/l NaCl, 4 g/l KHZPO4 adjusted to pH 7 .4 with NaOH. I
Precultures: A working stock of .DELTA.Steme-1 pPA102!CR4 was prepared from the seed culture by
streaking cells on an FA medimn plate containing 40 .mu.g/ml of kanamycin. A sweep from the
conuent growth zone on plate was cultured one time in liquid FA medium supplemented with
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kanamycin 40 .mu.g/ml to a nal O.D..sub.600nm of 4.0. This culture was checked for purity by
streaking on SBA plates, and diluted into multiple vials containing sterile 100% glycerol to a nal
glycerol concentration of 50% V/V!. These stocks were stored at -70.degree. C. A single vial was
removed at the start of each fermentation cycle and discarded after use. The defrosted cells were
streaked onto FA plates containing 40 .mu.g/ml kanamycin and incubated at least 16 hrs at 37.degree. C.
Aer 16 hrs the plated cells were used to inoculate 50 mls of FA medium supplemented with
40 .mu.g/ml kanamycin in a 250 ml bafed-Erlenmeyer ask Bellco Laboratories!. The culture was
incubated at 370.degree. C. at 200 rpm for 6 hrs or until an O.D..sub.600nm of 4-6 was obtained. The
cells were then subcultured into 50 mls of FA medium in an identical ask under identical conditions.
After 6 hrs, or a culture O.D..sub.600nm of 6.2-6.5, a 1.6% v/v! inoculum was transferred to 300 mls of
FA medium supplemented with 40 .mu.g/ml kanamycin in a 2 liter baffled Erlenmeyer and incubated at
37.degree. C. at 200 rpm for 7hrs, or until a nal O.D..sub.600nm of 3.5-3.7 was achieved. 0
Fermentation conditions: The fermentations described here were carried out using a New Brunswick
Bio-Flo 3000 equipped with a 5.0 liter working volume glass vessel and stainless steel headplate and
hemispherical bottom cooling dish. F our liters of FA medium were added to the vessel, which had been
previously completely disassembled, scrubbed in a dilute Envirochem solution and autoclaved for 15
min after the addition of 4 liters of H.sub.2 O. The polarographic DO.sub.2 probe Ingold! and pH
probes either liquid or gel lled, Ingold! were also inserted and all addition and sampling ports were
sealed or clamped and wrapped in aluminum foil. Addition lines consisted of surgical grade
autoclavable Tygon tubing Thomas Scientic! and all lines were sealed with the exception of the
condenser, which was left open to permit pressure release, but covered with aluminum foil. The vessel
was autoclaved using a 10 min exposure time at 121 .degree. C. and removed om the autoclave as soon
as sufficient cooling had occurred to allow opening of the autoclave. The vessel was then immediately
connected to the ferrnentor unit and the condenser line was connected to a sterile liquid trap and 0.2 .mu.
capsule lter to avoid the introduction of contaminants during the cooling process. The vessel was then
cooled to 37.degree. C. using the fermentor driven temperature control and positive pressure was
provided using compressed sterile ltered air. Once the vessel had cooled to 37.degree. C. sterile ltered
kanamycin was added to a nal concentration of 40 .mu.g/ml. The agitation was activated at 150 rpm
and aeration was adjusted to 1-1.2 volume/volume/min vvm! and antifoam C DOW!, that had been
diluted 10-fold into H.sub.2 O and autoclaved, was added to a nal concentration of 200 ppm.
A preinoculation sterility check was conducted for a of 16 hrs during which time pH,
agitation and temperature were continually monitored. After the 16 hrs required for DO.sub.2 probe
polarization, the DO.sub.2 was also monitored along with turbidity. The DO.sub.2 probe was calibrated
using an INGOLD calibration device which sets the zero value to 4 mA and 100% to the oxygen tension
determined by the solubility of oxygen in the medium after aeration and agitation at 37.degree. C. The
calibration and response of the electrode was then checked by sparging with pure N.sub.2. The vessel
was judged to be sterile if the pH and DO.sub.2 remained constant and no increase in turbidity was
observed. It should be emphasized that the short autoclave cycle for vessel sterilization was required to
minimize caramelization, Millard and other chemical degradation reactions which are problematic due
to the high concentrations of yeast extract and tryptone in FA medium. As an additional conrmation of
sterility, 50 mls was aseptically removed from the fermentor to a 250 mls Erlenmeyer and incubated at
37 .degree. C. at 200 rpm for 48 hrs with no sign of growth. Under the conditions outlined here
contamination has not been observed in more than 10 fermentation cycles.
Once the sterility of the vessel had been veried, the 300 ml inoculum described above was added to the
vessel through the addition port of the headplate and the initial O.D..sub.600nm was recorded. A sample
of the inoculum was also streaked on SBA plates and incubated for 48 hrs at 37 .degree. C. to verify
inoculum purity. Using the Bio-F lo 3000, aeration was maintained at 75% of saturation by increasing
agitation om the initial 150 rpm to a maximum of 400 rpm and ultimately by supplementing the 1 vvm
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aeration rate with pure oxygen. The mixture rate and percentages of air and oxygen were controlled by a
solenoid and algorithm developed by New Brunswick Scientic. Both gases had a working pressure of
approximately l0 psi.
The O.D..sub.600nm dry cell weight DCW!, production of PA, DO.sub.2, pH, agitation and
temperature were monitored throughout each fermentation cycle. The O.D..sub.600mn DCW and PA
production analysis were carried out by manually sampling the fermentation liquor at hourly intervals
using a sterile sampling port. O.D..sub.600mn was measured after dilution of the culture using sterile
medium prepared for that fermentation. For each O.D..sub.600nm determination, two appropriate
dilutions were made and results were considered acceptable only when both dilutions yielded a linear
response. DCWs were determined starting with a 2 hr point by centrifuging 10 mls of fermentation
liquor at 11,953 .times.g for 10 min, resuspending the cell pellet in 10 mls of sterile PBS and pelleting
the cells again under the same conditions. The cell pellet was resuspended in a minimal volume of PBS
and transferred quantitatively to a preweighted Eppendorf centrifuge tube and centrifuged at 14,000 rpm
for 5 min. Excess PBS was removed and the cell pellet was dried in a speed-vac for 72 hrs under
vacuum and a medium heat setting. A nal analysis of the dry weight versus O.D..sub.600nm revealed
that the relationship between the two parameters was adequately t with a linear function.
Fermentation Reproducibility: The reproducibility of the cell growth parameters, biomass and PA
production in fermentations carried out with the Bio-Flo 3000 under the conditions described above
have been summarized in Table I below. Two fermentations were carried out at 75% of the maximum
dissolved oxygen concentration in a strict batch mode with no pH control or additions other than
antifoam C. The variation in the agitation rate during the rst 100 min of the fermentation was the result
of the AGDO.sub.2 agitation DO.sub.2! control mode chosen to maintain the dissolved oxygen tension
at 7 5% of the maximmn. Briey, this algorithm attempts to control the oxygen tension by rst altering
the agitation rate until this proves insuicient, at which point the process air is supplemented with pure
oxygen as needed to maintain the desired DO.sub.2. The temperature was held constant at 37.degree.+/-
0.1.degree. C. The pH was monitored, but not regulated as an internal check on the aeration of the vessel
during the course of the fermentation. The fact that the pH revealed a decrease on only 0.2 pH units in
the rst 150 min was consistent with an aerobic culture metabolizing the limited carbohydrate supplied
with the yeast extract to CO.sub.2 and organic acids. Once the carbohydrate was exhausted after ca. 150
min, the bacillus switched to the utilization of amino acids and peptides for a carbon source, which
under aerobic conditions resulted in the release of NH.sub.4 OH and the observed increased culture pH.
These fermentations were sampled on an hourly basis and allowed to proceed until no further increase in
O.D..sub.600nm was observed over two time points. O.D..sub.600nm , DCW analysis and product
measurements were carried out for each sample as described above. Samples for PA production were
sterile ltered followed by the addition of HEPES and the complete protease cocktail as described under
PA quantitation. The samples were concentrated, desalted and ultimately concentrated 80-fold prior to
being analyzed using SDS-PAGE. The major band of the gel corresponded to the 83 kDa PA product.
An increasing in the intensity of the protein band was seen with increasing fermentation time. Study of a.
Westem blot of another time course of a batch fermentation was developed with polyclonal rabbit anti-
PA83. Comparison revealed that along with increasing PA 83 kDa there was also a pronounced increase
in the abundance and form of proteolytic degradation products of PA.
TABLE 1
Summary of Aerobic -DELTA.Sterne1 pPAlO2!CR4 Fermentations
Fermentation Final Conc. Final Yield Final Yield Specific Growth
Doubling Time
Conditions .mu.g PA83/ml mg PA83! mg PA83/g DCW! Rate
T.sub.D min!
Aerobic, Batch 51 235 8.10 0.0132 .
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min.sup.l 53
Aerobic, Batch 64
min.sup.l 51
Aerobic, Batch 45
min.sup.l 51
pH constant
Aerobic, FedBatch 68
min.sup.l 60
noncontinuous!
DCW = dry cell weight301 10.7 0.0136
225 7.40 0.0136
360 ND 0.0116
The data presented in Table 1 demonstrated that the PA yield on a unit volume and biomass basis, as
well as the cell growth parameters, were reproducible for the batch ferrnentations conducted without pH
control. The nal fermentation pH values of 8.57 and 8.67 after an elapsed fermentation time of ca. 8 hrs
were also comparable. The effect of prolonged exposure to these mildly alkaline conditions on cell
growth, PA production and subsequent degradation was investigated by repeating the fermentation at a
constant pH of 7.50+/-0.05 pH units. This was accomplished using the immersed vessel pH probe and
automated additions of 2 N HCI or 1 N NaOH. The results shown in Table 1 demonstrate that there was
no clear effect of constant pH on any of the parameters evaluated. SDS-PAGE analysis of the
fermentation time points sampled for PA production also revealed no signicant differences.
The nal fermentation presented in Table 1 was a noncontinuous fed-batch trial during which 1/ 10
volume of a 10-fold concentrate of sterile-ltered tryptone was added after 5 hrs or an O.D..sub.600mn
of 7.5. The result suggested that such fed-batch fermentations provide possible protocols for
improvement to increase yield and decrease proteolysis.
Harvest conditions: Fermentations were allowed to proceed until no further increase in O.D..sub.600nm
was observed. At this point, the fermentor was cooled to 10.degree. C. and the protease inhibitors
phenylmethylsulfonyl uoride PMSF!, 1,10-phenanthroline OP! and ethylenediamine tetraacetate
EDTA! were added to nal concentrations of 0.1, 0.05 and 2 mM, respectively. The cells were then
pumped om the fermentor vessel at room temperature using an Amicon DCI 0L concentrator equipped
with a 10-ft.sup.2 0.1 .mu. polysulfone hollow-ber cartridge. The fermentor liquor was diluted 1:1 with
25 mM diethanolamine DEA!, 50 mM NaCl, 2 mM EDTA, 0.1 mM PMSF adjusted to pH 8.9 with
HCl. The ltrate was collected at an operating pressure of less than 20 psi and transferred directly to a
second Amicon DC10L equipped with two 30 kDa cutoff 10-ft.sup.2 wound spiral cellulosic cartridges.
The ltrate was concentrated approximately 10-fold before being subjected to dialtration at an
operating pressure of less than 30 psi against the same buffer. The conductivity of the retentate was
monitored with an Amber Sciences conductivity meter and platinum immersion pencil-type electrode.
The dialtration step generally required 20 liters of buffer, but was considered complete only after the
conductivity of the concentrated retentate was equivalent to that of the starting buffer.
Quantitation of 83 kDa PA in crude fermentation liquor: The fermentation liquor was sampled using a
sterile port at regular intervals throughout the fermentation process. The samples for PA determination
were ltered through syringe type 0.2 .mu. cellulose acetate lters, 0.1 mM PMSF, 2 mM EDTA,
50 .mu.M OP and 20 mM HEPES pH7 .3 were added and the samples were frozen at -70.degree. C. The
samples were defrosted on ice and concentrated using Amicon Centricon 30 concentrators at
4500 .times.g. The samples were concentrated approximately 10-fold, diluted to the original volume
with 10 mM TRIS pH8.0, 0.1 mM PMSF, 2 mM EDTA, 0.05 .mu.M OP and concentrated again. The
concentrated sample was desalted again using the same buffer, frozen and fmally lyophilized using a
Speed-Vac. The dried samples were dissolved in 25 .mu.l of the TRIS buffer described above and
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diluted 1:1 with a 2.times.SDS solubilization buffer consisting of 50 mM Na.sub.2 CO.sub.3, 4% W/v!
SDS, 12% vlv! glycerol, 2% vlv! 2-mer-captoethanol and 0.01% W/v! Bromphenol Blue prior to
heating at 95.degree. C. for 5 min. The fermentation samples containing varying amounts of PA 83 kDa
were solubilized as described above and run on a Daiichi 4-20% gradient TRIS/TRICINE gel to
approximate total yield of PA. Two hundred to 2000 ng samples of puried PA were solubilized in the
same buffer and loaded onto the gel in constant total volume of 3 .mu.l. Three or four appropriate
dilutions of the fermentation samples determined from the rst gel were loaded onto the gel with the
standards and electrophoresed at 100 V initially and 140 V once the samples entered the separating gel
and until the Bromphenol Blue dye reached the bottom edge of the separating gel. The gel was then
xed in 10% v/v! acetic acid 20% vlv! MeOH for 10 min, rinsed with MQ H.sub.2 O and stained with
Coomassie Brilliant Blue 0.05% w/v! in 10% vlv! acetic acid for a minimum of 16 hrs to allow
complete and uniform staining. The stained gel was then destained in 10% vlv! acetic acid until the
backgrormd contained no visible residual dye. The gel was then scanned on a laser densitometer LKB,
Ultrascan XL Laser Densitometer!. Representative portions of the gel without protein were randomly
chosen and scanned to determine background absorption for an accurate baseline. The region to be
scanned for each lane containing PA was then visually aligned to insure that the entire protein peak and
adequate baseline were included in each scan. The scans were completed and the integration values were
determined using the LKB preprogrammed Gaussian algorithm and later were conrmed by cutting out
individual peaks and manually integrating based on peak weight. The resulting integration values were
plotted using Sigmaplot J andel!. Linear regression of the results revealed typical r values of 0.992-
0.996. The linear standard curve was then used to quantitate the amount of 83 kDa PA in the various
fermentation samples based on the same integration methods.
Purication: The exact volume and conductivity of the PA in DEA buffer was determined and solid KCI
was added to the solution to a nal concentration of 30 mM and conductivity of 10-11 mmhos/cm. The
PA was pumped with a peristaltic pump through a monoQ column prepared by collecting 100 mls of
hydrated Bio-Rad Macro Prep 50Q on a sintered glass lter and washing sequentially with 1 liter of 25
mM DEA, 50 mM NaCl, l mM EDTA, 50 .mu.M OP and 0.1 mM PMSF pH8.9 and 1 liter of the same
buffer with 30 mM KCI added. The conductivity 0-11 mrnhos/cm! and pH of 8.9 of the eluate om
the Macro Prep SOQ after the second wash were comparable to that of the PA solution after addition of
KCI. The Macro Prep SOQ resin was then degassed and slurry packed into a Pharmacia K column with a
Rainin Rabbit-Plus peristaltic pump at 48 rpm and a flow rate of 15 rnls/min. The fmal column volume
was .times.5 cm! 98 mls. The PA solution was pumped through the Macro Prep SOQ column at a rate
of 10 mls/min and the eluate was collected until all of the PA sample volume was loaded and the column
washed with an additional 100 mls of DEA/KCl buffer. The eluate containing unbound PA was
concentrated and dialtered using an 1-ft.sup.2 30 kDa cutoff cellulosic-Amicon wound spiral cartridge
at an operating pressure of 20 psi. The nal concentrate ca. 400 mls, 67 mmhos/cm! was passed
through a 0.2 A cellulose acetate lter. The ltered PA was loaded onto a Poros IIQ perfusion
chromatography column using a quaternary Waters 600E HPLC pump. The column was prepared by
hydrating seven grams of the Poros HQ perfusion resin in twice the packed bed volume of 2% w/v!
NaCl. After settling the resin was resuspended in six times the packed bed volume of 25 mM DEA pH
8.9, 50 mM NaCl, 7.5% v/v! ethylene glycol and allowed to settle overnight at room temperature. The
resin was then resuspended in three times the packed bed volume and nally in one and one-half times
the nal volume before the slurry was extensively degassed using a vacuum pump vacuum unknown!.
The entire degassed slurry was then transferred to a Waters AP 20.times. 1 00 mm glass HPLC column
and the column was packed in one step using the Waters 600E pumps at a flow rate of 20 mls/min and a
backpressure of 650 psi at room temperature. The column separation efficiency was then tested at a ow
rate of 10 rnls/min using a linear 1 M NaCl gradient and ovalbtunin 5 mg/ml Sigma! and bovine serum
albumin l0 mg/ml Sigma! in DEA as buffer as standard proteins. Approximately 100 mls of PA ca.
20-30 mg PA! cooled to 4-6.degree. C. was applied to re column and followed with a 20 min wash in
the starting buffer at room temperature to elute unbound material. The column was then developed with
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a linear gradient to 30% of the l M NaCl DEA elution buffer. The puried PA was found to elute
between 10- 15%, while the smaller molecular weight proteolytic breakdown products eluted as a
shoulder or partially resolved peak at 16-20% of the elution buffer. The resolution of the two peaks was
found to be a mction of content of PA proteolytic degradation products. The eluant was monitored at
280 nm and peak actions were collected by manual triggering of an ISCO fraction collector. Samples
of the peak actions were diluted into 5-10 volumes of TRIS pH8.0, 0.1 mM PMSF, 50 .mu.M OP, 1
mM EDTA buffer and concentrated using Amicon Centricon 30 concentrators at 4500.times. g at
4.degree. C. to approximately the initial sample volume. An equal volume of SDS-PAGE solubilization
buffer was added to the sample immediately prior to heating at 95.degree. C. for 5 min. Purity was
assessed from 8-25% SDS-PAGE PHAST gels Pharmacia! and actions with the highest purity were
combined and dialyzed against 40-50 volumes of 25 mM DEA pH8.9, 50 mM NaCl, 0.1 mM PMSF and
2 mM EDTA at 4.degree. C. for at least 16 hrs. Fractions judged empirically to be less than 95% pure
were rechromatographed under the same conditions and purity of the fractions was reassessed as
described above. All fractions of greater than 95% purity were ultimately combined, aliquoted and
frozen at -70.degree, C. subsequent to determination of the total PA concentration.
Analysis and characterization of puried 83 kDa PA: Puried PA was quantitated by measuring UV-
absorption at 280 nm using the relationship of 1 A.sub.280nm in a 1 cm pathlength cuvette is equals 1
mg PA/ml Leppla, 1988!. Results obtained in this manner were conrmed using the Bio-Rad Bradford
protein assay under conditions suggested by the manufacturer. PA purity was assessed using SDS-
PAGE under conditions described above. Capillary electrophoresis analytical assays have also proven
promising in the assessment of PA purity and amounts of residual protease inhibitors in nal product.
Feasibility studies using a 47 cm.times.50 .mu.m uncoated silica capillary and borate/SDS/acetonitrile
buffer revealed an excellent separation of the protein from residual protease inhibitors. Quantitation of
both protein and inhibitors has also proven possible, but the technique remains limited by the relatively
high limits of detection mM EDTA, 0.1 mM PMSF, and 0.05 mM OP! under current conditions.
Automated N-terminal sequencing was carried out with puried PA using an Applied Biosystems 470A
sequenator after desalting over Bio-Rad PDl 0 columns equilibrated with 5 mM NaCl and 1 mM
CaCl.sub.2. A unique N-terminal sequence was found and the rst six residues of the sequence were
identical to PA from the endogenous plasmid pX0l harbored by the USAMRIID B. antbracis Sterne
strain. In addition, the sequence corresponded exactly with the published DNA derived protein sequence
W elkos et al.!. Native gel electrophoresis under non-denaturing conditions revealed that PA puried
from .DELTA.Sterne-1 pPA102!CR4 also exhibited the microheterogeneity noted previously for PA
produced by the Steme strain. Cytotoxicity assays of the product using the macrophage lysis assay
Friedlander et al.! revealed that the titration curve of biological activity for PA from .DELTA.Sterne-1
pPA102!CR4 was indistinguishable om that generated for PA om the Sterne strain.
Evaluation of .DELTA.Sterne-1 pPAl02!CR4:
EXAMPLE 1
B. Anthracis .DELTA.Steme-1 pPAl02!CR4 was compared with its parent spore-forming strain B.
anthracis .DELTA.Steme-l pPA102!. Both organisms were plated onto sheep blood agar a preferred
medium for promoting bacterial spore production! and grown at 37 .degree. C. for 1 day, after which the
temperature was lowered to 25 .degree. C. for 4 days. The two strains were also grown in liquid
Leighton-Doi medium, which is designed to promote spore production, for 1 day at 37.degree. C.
followed by 4 days growth at 25 .degree. C. Growth from both agar and broth cultures were examined
under phase contrast microscopy for the presence of spores. Growth from all four cultures were then
resuspended in phosphate buffered saline to a concentration of about 10.sup.9 colony forming units
CFU! per ml. All four cultures were then heat shocked at 64.degree. C. for 60 minutes to kill vegetative
cells. Aliquots of 0.1 ml of the heat shocked material was then plated out onto sheep blood agar and
http://patft.uspt0.gov/netacgi/nph-Parser?Sectl=PTO2&Sect2=HIT0FF&u=/netahtml/sear... 6/28/2005
United States Patent: 6,316,006 Page 9 of l0
incubated at 37.degree. C. for 2 days.
Results:
B. anthracis .DELTA.Sterne~l pPAl 02!: Spores were seen under microscopic examination of material
from both the sheep blood agar cultures and the Leighton-Doi medium cultures. On sheep blood agar
plates containing heat shocked culture material om both sheep blood agar cultures and Leighton-Doi
medium cultures, there was conuent growth. The data clearly indicate that B.
anthracis .DELTA.Steme-l pPAl 02! forms spores.
B. anthracis A8tern-l pPA102!CR4: No spores were seen under microscopic examination of material
from both the sheep blood agar cultures and the Leighton-Doi medium cultures. On sheep blood agar
plates containing heat shocked cultures, there was no growth whatsoever. The data clearly indicate the
B. anthracis .DELTA.Sterne-l pPAl02!CR4, which has been deposited in the American Type Culture
Collection and has been assigned ATCC designation 69714, does not form spores.
EXAMPLE 2
B. anthracis .DELTA. Sterne-1 pPA102!CR4 was grown in an FA medium fermentor culture. No spores
were seen upon phase contract microscopic examination. Only medium-length and long chains of bacilli
were seen. Dilution plate counts on the culture determined that the culture contained l.86.times.l0.sup.9
CFU per ml. Three ml of culture was heat shocked at 60.degree. C. for 60 minutes, then 0.2 ml was
plated onto each of 5 plates of Tryptic soy agar. After incubation for 2 days at 37.degree. C., no colonies
were seen on the agar plates, indicating that spore production in the fennentor was less than l per
l.86.times. l0.sup.9 CFU. On two other fermentation runs with this strain, similar results were obtained.
No revertants to the parent spore-forming phenotype were observed.
The above process using an FA medium ferrnentor culture was repeated using the parent strain B.
anthracis .DELTA.Steme-l pPAl02!.
Growth on the tryptic soy agar after heat shock resulted in a total of 1000 total colonies, indicating that
lIl16 parent strain B. anthracis .DELTA.Steme-l pPAl02! had about 1000 spores per ml in the FA
medium, or 1 spore per 10.sup.6 CFU in the non-heat shocked medium.
EXAMPLE 3
Protective antigen PA! was prepared in accord with the teachings under Materials and Methods as
described above. The puried PA of B. anthracis .DELTA.Stern-1 pPAl02!CR4 was mixed in dierent
buffers phosphate buffered saline, HEPES, Tris, glycyl glycine GG!, sodium citrate; for example! and
combined with monophosphoryl lipid A MPL!, Squalene, Tween 80 and lecithin. The mixture was then
lyophilized. At 0 and 4 weeks, vials of lyophilized MPL/PA/emulsion were reconstituted in phosphate
buffered saline PBS! and injected in 0.5 ml doses containing 50 .mu.g of PA per dose. At l0 weeks, the
guinea pigs were aerosol challenged with approximately 36 medial lethal doses of virulent Bacillus
anthracis spores of the Ames strain. The following data shows status two weeks after the challenge.
Vaccine S/T* % Anti-PA**
PA in PBS + MPL emulsion! 10/12 83 29,427
PA in GG + MPL emulsion! 14/16 88 23,713
PA in Tris + MPL emulsion! 15/16 94 27,384
PA in HEPES + MPL emulsion! 15/15 100 25,482
PA in Citrate + MPL emulsion! 16/16 100 31,622
PBS O/4 O <10
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: United States Patent: 6,316,006 Page 10 of 10
-
- *Survived/Total, day 14 postchallenge
**Prechallenge serum titers to PA were determined by enzyme linked
immunosorbent assay. The geometric mean reciprocal titers were calculated
for each group and are expressed in this table.
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b7CALL INFORMATION CONTAINED
HEEIN I5 UNELASEIFIED
DATE l2l52000 BY 5032% UE B 22??-w?-222 93 6USAl92/IRI ID -92's~'92°92
l
The followin investigation was conducted by SpecialAgent between July 6 and 11, 2<><>5=
[Administrativez During 2001, BRUCE EDWARDS IVINS used
dialup America Online AOL! as his internet service provider
ISP!. Investigation to date has revealed that based on IVINS
home telephone number,[::::::::::::::] the AOL software would
have dialed three different numbers to connect to the internet.Two of the numbers| |and| I were leasedAOL by Genuity, Inc., and one was leased to AOL by UUNET, Inc.
On July 6, 2005, writer contacted.E::::::::::] UUNET,
Internet Inc., telephone number] lin order to obtain
'Protoc5l IP! information on BR ARDS IVINS for 2001.
[:::::]advised that if IVINS connected to the number operated by
UUNET to connect to AOL, they would have record of that
connection. [:::::]further advised that she would query UUNET's
databases to determine if IVINS telephone number had ever dialed
into UUNET's number.to
On July 11, 2005,[:::::]contacted writer and advised
that IVINS home number had never been used to connect with
UUNET . '
[:::::]did not provide any additional information.ANEDKETH
D
L I i
I I 4.
1
ALL IIJFIIIPIIJZTIIIIIIT CIIIIIITILIEEIJ n
279AWF-222936-USAMRIID 92'5<2>b HEPIEII-I LTi92ICLASSIFIEII~[:::::::::::] DATE l3l53UU BY 50324 UC B
1u
The followin investigation was conducted by SpecialAgent[IIIIIIIIIIIIIIIIl@n Ju1Y 8' 2°°5=
During the review of electronic evidence in support of
Major Case 184, draft agendas were found for the 4th
International Conference on Anthrax, held in Annapolis, Maryland
from June 10 thru 13, 2001. The conference was organized by
scientists from the United States Army Medical Research Instituteof Infectious Diseases USAMRIID!, namelyl |and BRQCE_y i
IY§NS.
Evidence review revealed two drafts of the agenda, one
labeled 4th Anthrax Wkshp.Scient.Prog#2 and the other labeled 4th
Anthrax Wkshp.Sci.Prog#4 dated 6/9/O0.
These agendas were compared to one another to determine
any changes made between drafts. The results of the comparison
is detailed below. Each noted difference will be labeled by the
section where the difference is found. The agendas will be
labeled Prog#2 and Prog#4. Copies of each agenda are attached to
and made part of this document. "z§*
Header I»
Prog#4 is dated 6/9/00 while Prog#2 is not dated.
Prog#4 includes an additional "REF."labeled 4th Anthrax
Wkshp.Lori#4.
Scientific Program section and "REF" section are in a
different order on Prog#4 than on Prog#2. 92
Day 1: Opening Remarks
Prog#4 listsl or USAMRIIDas the first speaker while Prog#2 lists or|[:::::] USAMRIID! as the first speaker.
Day 1: Section Two
Prog#4 header reads Anthrax: Threats and Risks while
Prog#2 reads Risks and hazards.
Prog# - - $0. other CDCre resentative: or api espoTse program POCioretc. . Prog#2 chairman is11.T.|IfDI-92§_.~ 'l"E"I
U.-.45 '
£~
279A-WF22293 6USAMRI ID
2b6
b7C
Day 1: Section Three
On Prog#4 under "Other methods for rapid detection!,
two additional items are listed than on Prog#2. These names areDiag. Sys. Div USAMRIID!; realtime PCR, Robotics a
Tox, USAMRIID! - flow cytometry titer determination.
Day 2: Section Five
Prog#4 header reads "Cell Structure and Function" while
Prog#2 reads "Structure and Function."
Day 2: Section Six
First subject in this section of Prog#4 reads "Plasmid
and chromosomal regulation of toxin gene expression" while the
first subjection of Prog#2 reads "The pag operon: Regulation of
PagA by PagR"
"Updates" section of Prog#4 includes a topic titled
"genetic constructs for improved expression of LF" while Prog#2
does not.
"Updates" section of Prog#2 includes a topic titled
"New candidate vaccines" while Prog#4 does not.
"The interaction of B. anthracis with macrophages"
section of Prog#4 includes topics titled "Macrophages in
pathogenesis of anthrax" and Effect of LF on cytokine production
by macrophages; USAMRIID! while Prog#2 does notProg#2 has a difierenf fills for this section, "The interaction
of B. anthracis with macrophages in pathogenesis."
Day 3: Section Seven
Header on Prog#4 is labeled "Immunity and Vaccines"
while Prog#2 is labeled "Vaccines and immune responses."
Prog#4 includes a presenter[:::::;]"under "Other
experimental vaccines" while Prog#2 does no .
Prog#2 includes the verbiage "possible subjects.."
after the topic titled "Correlates of immune protection" while
Prog#4 does not.
Day 3: Section Eight
I '
-r
b6
. b7C
279AWF222936USAMRIID
Q
Prog#4 includes a line item titled " "PA32":[::::;:] Los Alamos! under "toxin competitive inhibitors; while
Prog 2 does not.
Prog#4 assigns the section titled "Update on[Antiajiotics: efficacy, resistance lectures or posters!" to[::] USAMRIID while Prog#2 does not.
Additional Agenda
In addition to the abovementioned a endas, a
reliminarv agenda was written b nd sent toP |.BR.ucE lms for i ut and feedback. Based on a
comparison between Ehe preliminary agenda and Prog#2, very little
was changed. These same eight main topics were carried over from
the preliminary agenda to Prog#2. A copy of the preliminary
agenda is attached to and made part of this document.
: _v""_'A .. - ~, K - . ,-.,I. 1 = - e 1 ,_ .~- . . - ~ v_ .~...._,,_,-_t ,-1-,: -|_ l -';¢A..;_'?~; L: n~ ' ' y . . . 92, _. ,_ .1 I * »_, .- 74 .-:3 r.%>>'-92.'. £3: =~-- f-1 »"- <L,.Ti 92/rises; A --
b K
tile: 4th Anthrax Wksho.Sci.Proc|#4 6/9/00 bi; <3
4th international Conference on Anthrax
. ALL I]lIlFDRI-LILTECIIJ ClIlI92TTAII~TEDA . . . nnapOhs' MD U S A HEIREII-Il IS TEIIILLSSIFIEE
1O_13June 2001 DATE 12-1:5-some BY 50224 ur: stint,-*t-az,='rt+1
REF.: Files th Anthrax Wk viewers ! 4-th Anthrax Wkshp.people
! 4-th Anthrax Wkshp 4
Scientific Program [SUBJECT CATEGORIES and possible speakers and subjects]
DAY 1:
Opening Remarks ! USAMRHD!;
and? - ! [or present talk during the main
Conference Dinne : emerl us speake_r"]
#1: Ecology and Epidemiology
Chairman: | |
Anthrax and the internet: 15 min:
A balance between immediacy and accuracy of information
l:l2001 Global Anthrax Rerjort 15 min.
Updates - Foci and outbreaks of human anthrax possible lectures or posters!
1 Central Asian republics -
Russia
China ~
Updates: Foci and outbreaks in animals possible lectures or posters!
Canada/USA: domesticated and wild animals
'2Australia: Update
Africa:
South Africa?
Late-breakers
#2: Anthrax: Threats and Risks
Chairman: or other CDC representative:r Rapid Response program POC tc.!
Bioterrorism and the alleged use of anthrax:
Oven/iew and management
OR: Anthrax as a biological weapon:
Medical and Public Health Management _
[Working Group for Civilian Biotense:
iviitlpes, lilzase II PO15! I5 u-C-L 1 /yr
_____?______i_i V
, 92
Ftep tr CDO. or USAMRIID DOD -' no?! 136I 15-in
Disposal of stockpiles in the FSU:
discovery of abandoned spore stockpiles in Uzbekistan/Kazahkstan
[A Russian - sensitivity issue'?]
#3: Detection, Identication, and Classification of B. anthracis
Chairmanzl |
Genetic Diversity of B. anthracis
plasmi - ecific diversity:
pag gene diversity!
chromosomalpolymorphisms: i
t New markers and procedures '
MLVA/multiplex PCR!
vrrloci ol mor hisms
Genetic markers for detection: ,
|RAPD ngerprinting!
Updates on other enetic markers Lectures or Posters!
et al.-
SASP gene probes and Bacillus evolution!?
immunological detection: _Antibod -based systems of detection in natural samples -
NMRl ,
Monoclonal antibodies to spores and vegetative cells
Other methods for rapid detection Lectures or Posters!:
NNRl ' ribot ing DARPA!; molecular diagnostics and detectorsDiao. Sys. Div USAMRHD!; real-time POR, robotics, T ox, USAMRIID! - flow cytometry titer determination
Chairman's Overview: Identification and Diversity of B. anthracis
DAY 1 or 2:
Chairman:#4: l92/lole<|:ular Bioloqy and Genomics
l
The genome of B. anthracis strain Ames: sequence and analysis
lililam
Sequen 0 and pXO2: Updates
etc.!
Charace asmid replication sequences
DAY 2:
#5: Cell Structure and Function:
Chairman: IOII
Genetic Control of Sporulation and Germination OR
Genetc Control of S ore and Vegetative Cell Structure, <Revi@w=|-ii
Germination genes of B. anthracis: lreland, germination loci! A
group, germination loci!
Characteristics of the exos orium<updaie= ip
Spore Structural Proteins - [OR in Topic #3]
|:l
S-l_ayerHomolo ous Domains of bacterial surface roteins 9roup
#6: Genetic Regulation and Pathogenesis
Chairman:| I
Plasmid and chromosomal regulation of toxin gene expression
ld t'f' t f t -b d eniicai 0 recep or in in regions in PA two groups!: ,
- et al. ,
t l. e a
Oligom A and cellular uptake of toxin
Mechanism of anthrax toxin entry into cells
|:| ! Characterization of the PA Channel
! A polarized epithelial cell model for toxin entnl116
127C
r
Q - v
Role of Proteosome in LF toxicity
I Izt al.!
Updates lectures or posters!
LF Fusion -mediated delivery system, or
enetic constructs for improved expression of LF
| |et al.!
LF Mechanism of Action MAPKKs
The interaction of B. anthracis with macrophages:
Macro ha es in pathogenesis of anthrax
Effect of LF on cytokine production by macrophages:
USAMRHD! -
A"*"sp°/-be
137C
DAY3: _
- 136
Alternate: Combine categories #7 and #8: lmmu h laxis and Treatment 1°79@iaiim@i%
#7: lmmunit and Vaccines 'co- ~d 8- wins
AVA and rPA vaccine potency and safety: comparisons-in animals
lvins 01:
Efficacy against vaccine-refractory strains
or lvins!
DNA vaccines _
e I2
and t al.!
PA-producinp recombinant B.ant/vracis vaccines
LF-fusion proteins
Other experimental vaccines:
Correlates of immune protection
_ In vitro correlate/animal models
USAMRIID . -DERA CBD[:::::::] -
Human immune responses to anthrax vaccine -|:|
The SCID mouse model-
#8: u man Vaccines and Treatments]
Chairmen: -
Anthrax|vaccination:_ Safety and immiiinogenicity of alternate schedules and routes:
Surveill liia|ry personnel immunized with AVA:
Human|Clinical studies with PAVAX I
Human antitoxin ' '
"scFV": Los Alamos!
Toxin competitive inhibitors
0 g 6
106
SING Conference overview!: |7. 92/_ "PA32": Los Alamos! b
Update ' ics: efficacy, resistance lectures or posters!
- USAMRIID!
92Remarks
92
92
i
tile: 4th Anthrax Wksho.Scient.ProCl#2O
7 .
4th International-Conference on Anthrax
b6
Annapolis, MD U.S.A. b7C
10 - 13 June 2001
Scientific Program [SUBJECT CATEGORIES and possible speakers and subjects]
REF.: Files th Anthrax Wkshp.reviewers ! 4th Anthrax Wkshp.people
DAY 1:
Opening Remarks kUSAMRllD!;and? - ! [or present talk during the main
Conference Dinne : emeri us speaker"]
#1: Ecolo and Incidence
Chairmanzl |
Anthrax and the internet:15 min.A immediacy and accuracy of information _
2001 Global Anthrax Report
|:_|15 min.
Updates - Foci and outbreaks of human anthrax possible lectures or posters!
Central Asian republics
Russia
China
Updates: Foci and outbreaks in animals possible lectures or posters!
Canada/USA: domesticated and wild animals
Australia: Updatei
Africa" U dates- .||Namibia'?|:|South Africa?
Late-breakers
#2: Risks and hazardsChairman:| = A
Bioterrorism and the alleged use of anthrax:
Overview and management
OR: Anthrax as a biological weapon:
Medical and Public Health Management
[Working Group for Civilian Biofense:
Rep fro JHU, DHHS/CDC, OI USAMRIID DOD - I10?!lm I
/__ 35 tapes, Phase II PO15 ,Lvr'/rs! Qmcz . 53/
B
Disposal of stockpiles in the FSU:
_ discovery of abandoned spore stockpiles in Uzbekistan/Kazahkstan b6
[A Russian - sensitivity issue?] 1373
#3: Detection. Identification. and Epidemiology of B. anthracis
Chairman: I I
Genetic Diversity of B. anthracis _
plasmid-s ecitic diversity: '
pag gene diversity!
chromosomal polymorphisms:
New markers and procedures
MLVA/multiplex PCR!
vrrloci ol mor hisms
Genetic markers for detection:RAPD ' ' '
Update o e enetic markers Lectures or Posters!
SASP gene probes and Bacillus evolution!?
immunological detection:
Antibody-based f detection in natural samples
NMRl-
Monoclonal antibodies to spores and vegetative cells
|:|'"
Other methods tor ra id detection Lectures or Posters!
69 " ' pine
DARPA ' molecular dia ' !, gnostics and detectors
Chairman's Overview: Identification and Diversity of B. anthracis
DAY1or2: M
we
#4: Molecular Biology and Genomics
Chairmanzl |
The genome of B. anthracls strain Ames: sequence and analysis
liltlahl
Sequ
…[truncated]